Antisense oligonucleotide modulation of tumor necrosis factor-alpha (TNF-alpha) expression

ABSTRACT

Compositions and methods are provided for inhibiting the expression of human tumor necrosis factor-α (TNF-α). Antisense oligonucleotides targeted to nucleic acids encoding TNF-α are preferred. Methods of using these oligonucleotides for inhibition of TNF-α expression and for treatment of diseases, particularly inflammatory and autoimmune diseases, associated with overexpression of TNF-α are provided.

[0001] This application is a continuation-in-part of U.S. application Ser. No. 09/824,322, filed Apr. 2, 2001, which is a continuation-in part of allowed U.S. application Ser. No. 09/313,932, filed May 18, 1999 (U.S. Pat. No. 6,228,642), which is a continuation-in-part of U.S. application Ser. No. 09/166,186 filed Oct. 5, 1998 (U.S. Pat. No. 6,080,580).

FIELD OF THE INVENTION

[0002] This invention relates to compositions and methods for modulating expression of the human tumor necrosis factor-α (TNF-α) gene, which encodes a naturally present cytokine involved in regulation of immune function and implicated in infectious and inflammatory disease. This invention is also directed to methods for inhibiting TNF-α mediated immune responses; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the human TNF-α gene.

BACKGROUND OF THE INVENTION

[0003] Tumor necrosis factor a (TNF-α also cachectin) is an important cytokine that plays a role in host defense. The cytokine is produced primarily in macrophages and monocytes in response to infection, invasion, injury, or inflammation. Some examples of inducers of TNF-α include bacterial endotoxins, bacteria, viruses, lipopolysaccharide (LPS) and cytokines including GM-CSF, IL-1, IL-2 and IFN-γ.

[0004] TNFα interacts with two different receptors, TNF receptor I (TNFRI, p55) and TNFRII (p75), in order to transduce its effects, the net result of which is altered gene expression. Cellular factors induced by TNF-α include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-γ (IFN-γ), platelet derived growth factor (PDGF) and epidermal growth factor (EGF), and endothelial cell adhesion molecules including endothelial leukocyte adhesion molecule 1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (Tracey, K. J., et al., Annu. Rev. Cell Biol., 1993, 9, 317-343; Arvin, B., et al., Ann. NY Acad. Sci., 1995, 765, 62-71).

[0005] Despite the protective effects of the cytokine, overexpression of TNF-α often results in disease states, particularly in infectious, inflammatory and autoimmune diseases. This process may involve the apoptotic pathways (Ksontini, R., et al., J. Immunol., 1998, 160, 4082-4089). High levels of plasma TNFα have been found in infectious diseases such as sepsis syndrome, bacterial meningitis, cerebral malaria, and AIDS; autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease (including Crohn's disease), sarcoidosis, multiple sclerosis, Kawasaki syndrome, graft-versus-host disease and transplant (allograft) rejection; and organ failure conditions such as adult respiratory distress syndrome, congestive heart failure, acute liver failure and myocardial infarction (Eigler, A., et al., Immunol. Today, 1997, 18, 487-492). Other diseases in which TNFα is involved include asthma (Shah, A., et al., Clinical and Experimental Allergy, 1995, 25, 1038-1044), brain injury following ischemia (Arvin, B., et al., Ann. NY Acad. Sci., 1995, 765, 62-71), non-insulin-dependent diabetes mellitus (Hotamisligil et al., Science, 1993, 259, 87-90), insulin-dependent diabetes mellitus (Yang et al., J. Exp. Med., 1994, 180, 995-1004), hepatitis (Ksontini et al., J. Immunol., 1998, 160, 4082-4089), atopic dermatitis (Sumimoto et al., Arch. Dis. Child., 1992, 67, 277-279), and pancreatitis (Norman et al., Surgery, 1996, 120, 515-521). Further, inhibitors of TNF-α have been suggested to be useful for cancer prevention (Suganuma et al. (Cancer Res., 1996, 56, 3711-3715). Elevated TNF-α expression may also play a role in obesity (Kern, J. Nutr., 1997, 127, 1917S-1922S). TNF-α was found to be expressed in human adipocytes and increased expression, in general, correlated with obesity.

[0006] There are currently several approaches to inhibiting TNF-α expression. Approaches used to treat rheumatoid arthritis include a chimeric anti-TNF-α antibody, a humanized monoclonal anti-TNF-α antibody, and recombinant human soluble TNF-α receptor (Camussi, Drugs, 1998, 55, 613-620). Other examples are indirect TNF-α inhibitors including phosphodiesterase inhibitors (e.g., pentoxifylline) and metalloprotease inhibitors (Eigler et al., Immunol. Today, 1997, 18, 487-492). An additional class of direct TNF-α inhibitors is oligonucleotides, including triplex-forming oligonucleotides, ribozymes, and antisense oligonucleotides. Several publications describe the use of oligonucleotides targeting TNFα by non-antisense mechanisms. U.S. Pat. No. 5,650,316, WO 95/33493 and Aggarwal et al. (Cancer Research, 1996, 56, 5156-5164) disclose triplex-forming oligonucleotides targeting TNF-α. WO 95/32628 discloses triplex-forming oligonucleotides especially those possessing one or more stretches of guanosine residues capable of forming secondary structure. WO 94/10301 discloses ribozyme compounds active against TNF-α mRNA. WO 95/23225 discloses enzymatic nucleic acid molecules active against TNF-α mRNA.

[0007] A number of publications have described the use of antisense oligonucleotides targeting nucleic acids encoding TNF-α. The TNF-α gene has four exons and three introns. WO 93/09813 discloses TNF-α antisense oligonucleotides conjugated to a radioactive moiety, including sequences targeted to the 5′-UTR, AUG start site, exon 1, and exon 4 including the stop codon of human TNF-α. EP 0 414 607 B1 discloses antisense oligonucleotides targeting the AUG start codon of human TNF-α. WO 95/00103 claims antisense oligonucleotides to human TNF-α including sequences targeted to exon 1 including the AUG start site. Hartmann et al. (Mol. Med., 1996, 2, 429-438) disclose uniform phosphorothioates and mixed backbone phosphorothioate/phosphodiester oligonucleotides targeted to the AUG start site of human TNFα. Hartmann et al. (Antisense Nucleic Acid Drug Devel., 1996, 6, 291-299) disclose antisense phosphorothioate oligonucleotides targeted to the AUG start site, the exon 1/intron 1 junction, and exon 4 of human TNFα. d'Hellencourt et al. (Biochim. Biophys. Acta, 1996, 1317, 168-174) designed and tested a series of unmodified oligonucleotides targeted to the 5′-UTR, and exon 1, including the AUG start site, of human TNF-α. Additionally, one oligonucleotide each was targeted to exon 4 and the 3′-UTR of human TNF-α and one oligonucleotide was targeted to the AUG start site of mouse TNF-α. Rojanasakul et al. (J. Biol. Chem., 1997, 272, 3910-3914) disclose an antisense phosphorothioate oligonucleotide targeted to the AUG start site of mouse TNF-α. Taylor et al. (J. Biol. Chem., 1996, 271, 17445-17452 and Antisense Nucleic Acid Drug Devel., 1998, 8, 199-205) disclose morpholino, methyl-morpholino, phosphodiester and phosphorothioate oligonucleotides targeted to the 5′-UTR and AUG start codon of mouse TNF-α. Tu et al. (J. Biol. Chem., 1998, 273, 25125-25131) designed and tested 42 phosphorothioate oligonucleotides targeting sequences throughout the rat TNF-α gene.

[0008] Interestingly, some phosphorothioate oligodeoxynucleotides have been found to enhance lipopolysaccharide-stimulated TNF-α synthesis up to four fold due to nonspecific immunostimulatory effects (Hartmann et al. Mol. Med., 1996, 2, 429-438).

[0009] Accordingly, there remains an unmet need for therapeutic compositions and methods for inhibiting expression of TNF-α, and disease processes associated therewith.

SUMMARY OF THE INVENTION

[0010] The present invention provides oligonucleotides which are targeted to nucleic acids encoding TNF-α and are capable of modulating TNF-α expression. The present invention also provides chimeric oligonucleotides targeted to nucleic acids encoding human TNFα. The oligonucleotides of the invention are believed to be useful both diagnostically and therapeutically, and are believed to be particularly useful in the methods of the present invention.

[0011] The present invention also comprises methods of modulating the expression of human TNF-α in cells and tissues using the oligonucleotides of the invention. Methods of inhibiting TNF-α expression are provided; these methods are believed to be useful both therapeutically and diagnostically. These methods are also useful as tools, for example, for detecting and determining the role of TNFα in various cell functions and physiological processes and conditions and for diagnosing conditions associated with expression of TNF-α.

[0012] The present invention also comprises methods for diagnosing and treating infectious and inflammatory diseases, particularly diabetes, rheumatoid arthritis, Crohn's disease, pancreatitis, multiple sclerosis, atopic dermatitis and hepatitis using the oligonucleotides of the present invention. These methods are believed to be useful, for example, in diagnosing TNF-α-associated disease progression. These methods are believed to be useful both therapeutically, including prophylactically, and as clinical research and diagnostic tools.

[0013] One embodiment of the present invention is a method of treating an inflammatory disorder in an individual comprising administering to said individual an effective amount of an oligonucleotide up to 30 nucleotides in length complementary to a nucleic acid molecule encoding human tumor necrosis factor-α, wherein the oligonucleotide inhibits the expression of said human tumor necrosis factor-α and comprises at least an 8 nucleobase portion of SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 39, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 149, SEQ ID NO: 157, SEQ ID NO: 264, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 290, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 315, SEQ ID NO: 334, SEQ ID NO: 418, SEQ ID NO: 423, SEQ ID NO: 425, SEQ ID NO: 427, SEQ ID NO: 431, SEQ ID NO: 432, SEQ ID NO: 435, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 441, SEQ ID NO: 455, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 460, SEQ ID NO: 463, SEQ ID NO: 465, SEQ ID NO: 466, SEQ ID NO: 468, SEQ ID NO: 472, SEQ ID NO: 474, SEQ ID NO: 475, SEQ ID NO: 483, SEQ ID NO: 485, SEQ ID NO: 494 or SEQ ID NO: 496. Preferably, the antisense oligonucleotide is administered orally. In one aspect of this preferred embodiment, the inflammatory disorder is inflammatory bowel disease, Crohn's disease, colitis or rheumatoid arthritis. Preferably, the oligonucleotide comprises at least one modified intersugar linkage. Preferably, the modified intersugar linkage is a phosphorothioate or methylene(methylimino) intersugar linkage. In another aspect of this preferred embodiment, the oligonucleotide comprises at least one 2′-O-methoxyethyl modification. Preferably, the oligonucleotide comprises at least one 5-methyl cytidine. In one aspect of this preferred embodiment, every cytidine residue is a 5-methyl cytidine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGS. 1A-B are graphs showing collagen-induced arthritis (CIA) onset as determined by percent incidence in mice. Incidence=number of mice with at least one affected paw/total number of mice per group. FIG. 1A shows the effect of low dose range of ISIS 25302 anti-TNF-α antisense oligonucleotide in comparison to treatment by an anti-TNF-α mAb. FIG. 1B shows the effect of high dose range treatment by ISIS 25302 in comparison to treatment by an 8 mismatch control oligonucleotide (ISIS 30782).

[0015]FIG. 2 is a graph showing “total” histological scores for colon tissue from IL-10^(−/−) mice treated with saline (vehicle), ISIS 25302 or 8MM Con. As recorded in Table 27. Results are expressed as mean ″ standard deviation (n=6). The asterisk indicates a significant difference (p<0.05) in comparison to the vehicle group.

[0016] FIGS. 3A-B show the basal (FIG. 3A) and LPS-induced (FIG. 3B) levels of TNF-α secretion from colon tissue of IL-10^(−/−) mice post-treatment with ISIS 25302 and the 8 base mismatch control oligonucleotide 30782 (8MM). Doses of oligonucleotide are shown in parentheses (mg/kg). Secretion levels (pg/gm-tissue) are shown in the y-axis. The mean values ″ standard deviation (n=7 to 9) are shown.

[0017] FIGS. 4A-B show the basal (FIG. 4A) and LPS-induced (FIG. 4B) levels of IFN-γ secretion from colon tissue of IL-10^(−/−) mice post-treatment with ISIS 25302 and the 8 base mismatch control oligonucleotide 30782 (8MM). Doses of oligonucleotide are shown in parentheses (mg/kg). Secretion levels (pg/gm-tissue) are shown in the y-axis. The mean values ″ standard deviation (n=6 to 9) are shown.

[0018] FIGS. 5A-B show the efficacy of ISIS 25302 versus anti-mouse TNF-α mAb in the acute model of DSS-induced colitis. FIG. 5A shows the disease activity index (DAI). FIG. 5B shows the effect of different treatments on colon length. Results are expressed as the mean ″ S.E.M., where n=7. Asterisks show a significant difference from saline treated (*) or normal (*′) group (p<0.05).

[0019] FIGS. 6A-B show that the prevention of acute colitis by ISIS 25302 in the DSS-induced colitis molecule is sequence-dependent. FIG. 5A shows DAI versus treatment. FIG. 5B shows the effect of different treatments on colon length. Asterisks indicate significant differences from saline (*) or 1.0 mg/kg 8MM Con (*″) treated group (p<0.05).

[0020] FIGS. 6A-B are graphs showing the efficacy of ISIS 25302 in the DSS-induced mouse model of chronic colitis based on DAI. FIG. 6A shows the mean DAI of each group over the course of the two cycle DSS-induced chronic colitis study. FIG. 6B shows the mean DAI at representative cycle times. The doses are indicated in parentheses (mg/kg). Results are expressed as the mean S.E.M., where n=8 to 10. Asterisks indicate statistical significance in comparison to the Vehicle group (P<0.05).

[0021] FIGS. 8A-B show histopathology of colon tissue from mice administered DSS in the two cycle chronic colitis model. Results are expressed as mean S.E.M. FIG. 8A shows the total inflammation and crypt scores. Acute inflammatory infiltrates consist of granulocytes, lymphocytes and plasma cells. Chronic inflammatory infiltrates consist of granulocytes, lymphocytes, plasma cells, monocytes and macrophages. FIG. 8B shows histological scores of different regions of the colon. PA=proximal acute inflammation score, DA=distal acute inflammation score, PC=proximal chronic inflammation score, DC=distal chronic inflammation score, PCS=proximal crypt score and DCS=distal crypt score. Asterisks indicate statistical significance in comparison to the Vehicle group (p<0.05).

[0022]FIG. 9 shows TNFα mRNA levels from longitudinal sections of colon tissue derived from each mouse at time of sacrifice in the chronic colitis model (mean S.E.M.). Group A=0.25 mg/kg ISIS 25302, group B=Vehicle, group C=anti-TNF mAb, group D=no treatment, group E=2.5 mg/kg ISIS 25302, group F=12.5 mg/kg ISIS 25302.

DETAILED DESCRIPTION OF THE INVENTION

[0023] TNF-α plays an important regulatory role in the immune response to various foreign agents. Overexpression of TNFα results in a number of infectious and inflammatory diseases. As such, this cytokine represents an attractive target for treatment of such diseases. In particular, modulation of the expression of TNFα may be useful for the treatment of diseases such as Crohn's disease, diabetes mellitus, multiple sclerosis, rheumatoid arthritis, hepatitis, pancreatitis and asthma.

[0024] The present invention employs antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding TNF-α, ultimately modulating the amount of TNF-α produced. This is accomplished by providing oligonucleotides which specifically hybridize with nucleic acids, preferably mRNA, encoding TNFα.

[0025] This relationship between an antisense compound such as an oligonucleotide and its complementary nucleic acid target, to which it hybridizes, is commonly referred to as “antisense”. “Targeting” an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a foreign nucleic acid from an infectious agent. In the present invention, the targets are nucleic acids encoding TNF-α; in other words, a gene encoding TNF-α, or mRNA expressed from the TNF-α gene. mRNA which encodes TNF-α is presently the preferred target. The targeting process also includes determination of a site or sites within the nucleic acid sequence for the antisense interaction to occur such that modulation of gene expression will result.

[0026] In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5′-untranslated region, the 3′-untranslated region, the 5′ cap region and intron/exon junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides. The oligonucleotide may therefore be specifically hybridizable with a transcription initiation site region, a translation initiation codon region, a 5′ cap region, an intron/exon junction, coding sequences, a translation termination codon region or sequences in the 5′- or 3′-untranslated region. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon.” A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding TNF-α, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region,” “AUG region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. This region is a preferred target region. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. This region is a preferred target region. The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other preferred target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

[0027] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a pre-mRNA transcript to yield one or more mature mRNAs. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., exon-exon or intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. Targeting particular exons in alternatively spliced mRNAs may also be preferred. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

[0028] Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.

[0029] “Hybridization”, in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them.

[0030] “Specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarily such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide.

[0031] It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarily to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.

[0032] Hybridization of antisense oligonucleotides with mRNA interferes with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.

[0033] The overall effect of interference with mRNA function is modulation of expression of TNF-α. In the context of this invention “modulation” means either inhibition or stimulation; i.e., either a decrease or increase in expression. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression, or reverse transcriptase PER, as taught in the examples of the instant application or by Western blot or ELIZA assay of protein expression, or by an immunoprecipitation assay of protein expression. Effects of antisense oligonucleotides of the present invention on TNF-α expression can also be determined as taught in the examples of the instant application. Inhibition is presently a preferred form of modulation.

[0034] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.

[0035] The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697). The use of these dsRNAs targeted to nucleic acid encoding TNFα is also within the scope of the present invention. These dsRNAs target the same or similar regions to those targeted by antisense oligonucleotides.

[0036] The oligonucleotides of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the oligonucleotide. For example, if the first nucleotide is an adenosine, variants may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the oligonucleotide. These oligonucleotides are then tested using the methods described herein to determine their ability to inhibit expression of TNF-α.

[0037] The oligonucleotides of this invention can be used in diagnostics, therapeutics, prophylaxis, and as research reagents and in kits. Since the oligonucleotides of this invention hybridize to nucleic acids encoding TNFα, sandwich, calorimetric and other assays can easily be constructed to exploit this fact. Provision of means for detecting hybridization of oligonucleotides with the TNFα gene or mRNA can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of TNFα may also be prepared.

[0038] The present invention is also suitable for diagnosing abnormal inflammatory states in tissue or other samples from patients suspected of having an inflammatory disease such as rheumatoid arthritis. The ability of the oligonucleotides of the present invention to inhibit inflammatory processes may be employed to diagnose such states. A number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue sample with an oligonucleotide of the invention under conditions selected to permit detection and, usually, quantitation of such inhibition. In the context of this invention, to “contact” tissues or cells with an oligonucleotide or oligonucleotides means to add the oligonucleotide(s), usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the oligonucleotide(s) to cells or tissues within an animal.

[0039] The oligonucleotides of this invention may also be used for research purposes. Thus, the specific hybridization exhibited by the oligonucleotides may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art.

[0040] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence of nucleases.

[0041] The antisense compounds in accordance with this invention preferably comprise from about 5 to about 50 nucleobases. Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e., from about 8 to about 30 linked nucleosides). As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidine. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0042] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0043] Oligomer and Monomer Modifications

[0044] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside linkage or in conjunction with the sugar ring the backbone of the oligonucleotide. The normal internucleoside linkage that makes up the backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0045] Modified Internucleoside Linkages

[0046] Specific examples of preferred antisense oligomeric compounds useful in this invention include oligonucleotides containing modified e.g. non-naturally occurring internucleoside linkages. As defined in this specification, oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0047] In the C. elegans system, modification of the internucleotide linkage (phosphorothioate) did not significantly interfere with RNAi activity. Based on this observation, it is suggested that certain preferred oligomeric compounds of the invention can also have one or more modified internucleoside linkages. A preferred phosphorus containing modified internucleoside linkage is the phosphorothioate internucleoside linkage.

[0048] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0049] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0050] In more preferred embodiments of the invention, oligomeric compounds have one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester internucleotide linkage is represented as —O—P(═O)(OH)—O—CH₂—]. The MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Preferred amide internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,602,240.

[0051] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0052] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0053] Oligomer Mimetics

[0054] Another preferred group of oligomeric compounds amenable to the present invention includes oligonucleotide mimetics. The term mimetic as it is applied to oligonucleotides is intended to include oligomeric compounds wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with novel groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA oligomeric compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA oligomeric compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA oligomeric compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0055] PNA has been modified to incorporate numerous modifications since the basic PNA structure was first prepared. The basic structure is shown below:

[0056] wherein

[0057] Bx is a heterocyclic base moiety;

[0058] T₄ is hydrogen, an amino protecting group, —C(O)R₅, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group, a reporter group, a conjugate group, a D or L α-amino acid linked via the α-carboxyl group or optionally through the ω-carboxyl group when the amino acid is aspartic acid or glutamic acid or a peptide derived from D, L or mixed D and L amino acids linked through a carboxyl group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

[0059] T₅ is —OH, —N(Z₁)Z₂, R₅, D or L α-amino acid linked via the α-amino group or optionally through the ω-amino group when the amino acid is lysine or ornithine or a peptide derived from D, L or mixed D and L amino acids linked through an amino group, a chemical functional group, a reporter group or a conjugate group;

[0060] Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

[0061] Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group, —C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the α-carboxyl group or optionally through the ω-carboxyl group when the amino acid is aspartic acid or glutamic acid or a peptide derived from D, L or mixed D and L amino acids linked through a carboxyl group;

[0062] Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl, —C(═O)—CH₃, benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;

[0063] each J is O, S or NH;

[0064] R₅ is a carbonyl protecting group; and

[0065] n is from 2 to about 50.

[0066] Another class of oligonucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. A preferred class of linking groups have been selected to give a non-ionic oligomeric compound. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based oligomeric compounds are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. The morpholino class of oligomeric compounds have been prepared having a variety of different linking groups joining the monomeric subunits.

[0067] Morpholino nucleic acids have been prepared having a variety of different linking groups (L₂) joining the monomeric subunits. The basic formula is shown below:

[0068] wherein

[0069] vT₁ is hydroxyl or a protected hydroxyl;

[0070] T₅ is hydrogen or a phosphate or phosphate derivative;

[0071] L₂ is a linking group; and

[0072] n is from 2 to about 50.

[0073] A further class of oligonucleotide mimetic is referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in an DNA/RNA molecule is replaced with a cyclohenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation. Furthermore the incorporation of CeNA into a sequence targeting RNA was stable to serum and able to activate E. Coli RNase resulting in cleavage of the target RNA strand.

[0074] The general formula of CeNA is shown below:

[0075] wherein

[0076] each Bx is a heterocyclic base moiety;

[0077] T₁ is hydroxyl or a protected hydroxyl; and

[0078] T₂ is hydroxyl or a protected hydroxyl.

[0079] Another class of oligonucleotide mimetic (anhydrohexitol nucleic acid) can be prepared from one or more anhydrohexitol nucleosides (see, Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) and would have the general formula:

[0080] A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C, 4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNA analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10 C), stability towards 3′-exonucleolytic degradation and good solubility properties. The basic structure of LNA showing the bicyclic ring system is shown below:

[0081] The conformations of LNAs determined by 2D NMR spectroscopy have shown that the locked orientation of the LNA nucleotides, both in single-stranded LNA and in duplexes, constrains the phosphate backbone in such a way as to introduce a higher population of the N-type conformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53). These conformations are associated with improved stacking of the nucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18, 1365-1370).

[0082] LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNA hybridization was shown to be the most thermally stable nucleic acid type duplex system, and the RNA-mimicking character of LNA was established at the duplex level. Introduction of 3 LNA monomers (T or A) significantly increased melting points (Tm=+15/+11) toward DNA complements. The universality of LNA-mediated hybridization has been stressed by the formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking of LNA was reflected with regard to the N-type conformational restriction of the monomers and to the secondary structure of the LNA:RNA duplex.

[0083] LNAs also form duplexes with complementary DNA, RNA or LNA with high thermal affinities. Circular dichroism (CD) spectra show that duplexes involving fully modified LNA (esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination of an LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer. Recognition of double-stranded DNA has also been demonstrated suggesting strand invasion by LNA. Studies of mismatched sequences show that LNAs obey the Watson-Crick base pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands.

[0084] Novel types of LNA-oligomeric compounds, as well as the LNAs, are useful in a wide range of diagnostic and therapeutic applications. Among these are antisense applications, PCR applications, strand-displacement oligomers, substrates for nucleic acid polymerases and generally as nucleotide based drugs. Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638.) The authors have demonstrated that LNAs confer several desired properties to antisense agents. LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. LNA/DNA copolymers exhibited potent antisense activity in assay systems as disparate as G-protein-coupled receptor signaling in living rat brain and detection of reporter genes in Escherichia coli. Lipofectin-mediated efficient delivery of LNA into living human breast cancer cells has also been accomplished.

[0085] The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

[0086] The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., PCT International Application WO 98-DK393 19980914). Furthermore, synthesis of 2′-amino-LNA, a novel conformationally restricted high-affinity oligonucleotide analog with a handle has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-Amino- and 2′-methylamino-LNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

[0087] Further oligonucleotide mimetics have been prepared to include bicyclic and tricyclic nucleoside analogs having the formulas (amidite monomers shown):

[0088] (see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberg et al., J. Am. Chem. Soc., 2002, 124, 5993-6002). These modified nucleoside analogs have been oligomerized using the phosphoramidite approach and the resulting oligomeric compounds containing tricyclic nucleoside analogs have shown increased thermal stabilities (Tm's) when hybridized to DNA, RNA and itself. Oligomeric compounds containing bicyclic nucleoside analogs have shown thermal stabilities approaching that of DNA duplexes.

[0089] Another class of oligonucleotide mimetic is referred to as phosphonomonoester nucleic acids incorporate a phosphorus group in a backbone the backbone. This class of olignucleotide mimetic is reported to have useful physical and biological and pharmacological properties in the areas of inhibiting gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex-forming oligonucleotides), as probes for the detection of nucleic acids and as auxiliaries for use in molecular biology.

[0090] The general formula (for definitions of Markush variables see: U.S. Pat. Nos. 5,874,553 and 6,127,346 herein incorporated by reference in their entirety) is shown below.

[0091] Another oligonucleotide mimetic has been reported wherein the furanosyl ring has been replaced by a cyclobutyl moiety.

[0092] Modified Sugars

[0093] Oligomeric compounds of the invention may also contain one or more substituted sugar moieties. Preferred oligomeric compounds comprise a sugar substituent group selected from: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N— alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise a sugar substituent group selected from: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-aminoethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂.

[0094] Other preferred sugar substituent groups include methoxy (—O—CH₃), aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂) , O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-Sugar substituent groups may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0095] Further representative sugar substituent groups include groups of formula I_(a) or II_(a):

[0096] wherein:

[0097] R_(b) is O, S or NH;

[0098] R_(d) is a single bond, O, S or C(═O);

[0099] R_(e) is C₁-C₁₀ alkyl, N(R_(k)) (R_(m)), N(R_(k)) (R_(n)), N═C(R_(p)) (R_(q)), N═C(R_(p)) (R_(r)) or has formula III_(a);

[0100] R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;

[0101] R_(r) is —R_(x)—R_(y);

[0102] each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen, C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

[0103] or optionally, R_(u) and R_(v), together form a phthalimido moiety with the nitrogen atom to which they are attached;

[0104] each R_(w) is, independently, substituted or unsubstituted C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, isobutyryl, phenyl or aryl;

[0105] R_(k) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

[0106] R_(p) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

[0107] R_(x) is a bond or a linking moiety;

[0108] R_(y) is a chemical functional group, a conjugate group or a solid support medium;

[0109] each R_(m) and R_(n) is, independently, H, a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH₃ ⁺, N (R_(u))(R_(v)), guanidino and acyl where said acyl is an acid amide or an ester;

[0110] or R_(m) and R_(n), together, are a nitrogen protecting group, are joined in a ring structure that optionally includes an additional heteroatom selected from N and O or are a chemical functional group;

[0111] R_(i) is OR_(z), SR_(z), or N(R_(z))₂;

[0112] each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);

[0113] R_(f), R_(g) and R_(h) comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;

[0114] R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R_(k))(R_(m)) OR_(k), halo, SR_(k) or CN;

[0115] m_(a) is 1 to about 10;

[0116] each mb is, independently, 0 or 1;

[0117] mc is 0 or an integer from 1 to 10;

[0118] md is an integer from 1 to 10;

[0119] me is from 0, 1 or 2; and

[0120] provided that when mc is 0, md is greater than 1.

[0121] Representative substituents groups of Formula I are disclosed in U.S. patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled “Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in its entirety.

[0122] Representative cyclic substituent groups of Formula II are disclosed in U.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998, entitled “RNA Targeted 2′-Oligomeric compounds that are Conformationally Preorganized,” hereby incorporated by reference in its entirety.

[0123] Particularly preferred sugar substituent groups include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.

[0124] Representative guanidino substituent groups that are shown in formula III and IV are disclosed in co-owned U.S. patent application Ser. No. 09/349,040, entitled “Functionalized Oligomers”, filed Jul. 7, 1999, hereby incorporated by reference in its entirety.

[0125] Representative acetamido substituent groups are disclosed in U.S. Pat. No. 6,147,200 which is hereby incorporated by reference in its entirety.

[0126] Representative dimethylaminoethyloxyethyl substituent groups are disclosed in International Patent Application PCT/US99/17895, entitled “2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6, 1999, hereby incorporated by reference in its entirety.

[0127] Modified Nucleobases/Naturally Occurring Nucleobases

[0128] Oligomeric compounds may also include nucleobase (often referred to in the art simply as “base” or “heterocyclic base moiety”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases also referred herein as heterocyclic base moieties include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0129] Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0130] In one aspect of the present invention oligomeric compounds are prepared having polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G-clamps or cytidine analogs. Many of these polycyclic heterocyclic compounds have the general formula:

[0131] Representative cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀=O, R₁₁-R₁₄=H) [Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16, 1837-1846], 1,3-diazaphenothiazine-2-one (R₁₀=S, R₁₁-R₁₄=H), [Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874] and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀=O, R₁₁-R₁₄=F) [Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388]. Incorporated into oligonucleotides these base modifications were shown to hybridize with complementary guanine and the latter was also shown to hybridize with adenine and to enhance helical thermal stability by extended stacking interactions(also see U.S. patent application entitled “Modified Peptide Nucleic Acids” filed May 24, 2002, Ser. No. 10/155,920; and U.S. patent application entitled “Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser. No. 10/013,295, both of which are commonly owned with this application and are herein incorporated by reference in their entirety).

[0132] Further helix-stabilizing properties have been observed when a cytosine analog/substitute has an aminoethoxy moiety attached to the rigid 1,3-diazaphenoxazine-2-one scaffold (R₁₀=O, R₁₁=—O—(CH₂)₂—NH₂, R₁₂₋₁₄=H) [Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532]. Binding studies demonstrated that a single incorporation could enhance the binding affinity of a model oligonucleotide to its complementary target DNA or RNA with a ΔT_(m) of up to 18° relative to 5-methyl cytosine (dC5^(me)), which is the highest known affinity enhancement for a single modification, yet. On the other hand, the gain in helical stability does not compromise the specificity of the oligonucleotides. The T_(m) data indicate an even greater discrimination between the perfect match and mismatched sequences compared to dC5^(me). It was suggested that the tethered amino group serves as an additional hydrogen bond donor to interact with the Hoogsteen face, namely the O6, of a complementary guanine thereby forming 4 hydrogen bonds. This means that the increased affinity of G-clamp is mediated by the combination of extended base stacking and additional specific hydrogen bonding.

[0133] Further tricyclic heterocyclic compounds and methods of using them that are amenable to the present invention are disclosed in U.S. Pat. No. 6,028,183, which issued on May 22, 2000, and U.S. Pat. No. 6,007,992, which issued on Dec. 28, 1999, the contents of both are commonly assigned with this application and are incorporated herein in their entirety.

[0134] The enhanced binding affinity of the phenoxazine derivatives together with their uncompromised sequence specificity makes them valuable nucleobase analogs for the development of more potent antisense-based drugs. In fact, promising data have been derived from in vitro experiments demonstrating that heptanucleotides containing phenoxazine substitutions are capable to activate RNaseH, enhance cellular uptake and exhibit an increased antisense activity [Lin, K-Y; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532]. The activity enhancement was even more pronounced in case of G-clamp, as a single substitution was shown to significantly improve the in vitro potency of a 20mer 2′-deoxyphosphorothioate oligonucleotides [Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518]. Nevertheless, to optimize oligonucleotide design and to better understand the impact of these heterocyclic modifications on the biological activity, it is important to evaluate their effect on the nuclease stability of the oligomers.

[0135] Further modified polycyclic heterocyclic compounds useful as heterocyclcic bases are disclosed in but not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. No. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. patent application Ser. No. 09/996,292 filed Nov. 28, 2001, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0136] The oligonucleotides of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the oligonucleotide. For example, if the first nucleotide is an adenosine, variants may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the oligonucleotide. Thus, a 20-mer may comprise 60 variations (20 positions×3 alternates at each position) in which the original nucleotide is substituted with any of the three alternate nucleotides. These oligonucleotides are then tested using the methods described herein to determine their ability to inhibit expression of HCV mRNA and/or HCV replication.

[0137] Conjugates

[0138] A further preferred substitution that can be appended to the oligomeric compounds of the invention involves the linkage of one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting oligomeric compounds. In one embodiment such modified oligomeric compounds are prepared by covalently attaching conjugate groups to functional groups such as hydroxyl or amino groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937.

[0139] The oligomeric compounds of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0140] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0141] Chimeric Oligomeric Compounds

[0142] It is not necessary for all positions in an oligomeric compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligomeric compound or even at a single monomeric subunit such as a nucleoside within a oligomeric compound. The present invention also includes oligomeric compounds which are chimeric oligomeric compounds. “Chimeric” oligomeric compounds or “chimeras,” in the context of this invention, are oligomeric compounds that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a nucleic acid based oligomer.

[0143] Chimeric oligomeric compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligomeric compound may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligomeric compounds when chimeras are used, compared to for example phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0144] Chimeric oligomeric compounds of the invention may be formed as composite structures of two or more oligonucleotides, oligonucleotide analogs, oligonucleosides and/or oligonucleotide mimetics as described above. Such oligomeric compounds have also been referred to in the art as hybrids hemimers, gapmers or inverted gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0145] 3′-endo Modifications

[0146] In one aspect of the present invention oligomeric compounds include nucleosides synthetically modified to induce a 3′-endo sugar conformation. A nucleoside can incorporate synthetic modifications of the heterocyclic base, the sugar moiety or both to induce a desired 3′-endo sugar conformation. These modified nucleosides are used to mimic RNA like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3′-endo conformational geometry. There is an apparent preference for an RNA type duplex (A form helix, predominantly 3′-endo) as a requirement (e.g. trigger) of RNA interference which is supported in part by the fact that duplexes composed of 2′-deoxy-2′-F-nucleosides appears efficient in triggering RNAi response in the C. elegans system. Properties that are enhanced by using more stable 3′-endo nucleosides include but aren't limited to modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage. The present invention provides oligomeric triggers of RNAi having one or more nucleosides modified in such a way as to favor a C3′-endo type conformation.

[0147] Nucleoside conformation is influenced by various factors including substitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2′ position to favor the 3′-endo conformation can be achieved while maintaining the 2′-OH as a recognition element, as illustrated in FIG. 2, below (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference for the 3′-endo conformation can be achieved by deletion of the 2′-OH as exemplified by 2′deoxy-2° F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3′-endo conformation positioning the electronegative fluorine atom in the axial position. Other modifications of the ribose ring, for example substitution at the 4′-position to give 4′-F modified nucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example modification to yield methanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) also induce preference for the 3′-endo conformation. Along similar lines, oligomeric triggers of RNAi response might be composed of one or more nucleosides modified in such a way that conformation is locked into a C3′-endo type conformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.) Examples of modified nucleosides amenable to the present invention are shown below in Table I. These examples are meant to be representative and not exhaustive. TABLE I

[0148] The preferred conformation of modified nucleosides and their oligomers can be estimated by various methods such as molecular dynamics calculations, nuclear magnetic resonance spectroscopy and CD measurements. Hence, modifications predicted to induce RNA like conformations, A-form duplex geometry in an oligomeric context, are selected for use in the modified oligoncleotides of the present invention. The synthesis of numerous of the modified nucleosides amenable to the present invention are known in the art (see for example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988, Plenum press., and the examples section below.) Nucleosides known to be inhibitors/substrates for RNA dependent RNA polymerases (for example HCV NS5B

[0149] In one aspect, the present invention is directed to oligonucleotides that are prepared having enhanced properties compared to native RNA against nucleic acid targets. A target is identified and an oligonucleotide is selected having an effective length and sequence that is complementary to a portion of the target sequence. Each nucleoside of the selected sequence is scrutinized for possible enhancing modifications. A preferred modification would be the replacement of one or more RNA nucleosides with nucleosides that have the same 3′-endo conformational geometry. Such modifications can enhance chemical and nuclease stability relative to native RNA while at the same time being much cheaper and easier to synthesize and/or incorporate into an oligonulceotide. The selected sequence can be further divided into regions and the nucleosides of each region evaluated for enhancing modifications that can be the result of a chimeric configuration. Consideration is also given to the 5′ and 3′-termini as there are often advantageous modifications that can be made to one or more of the terminal nucleosides. The oligomeric compounds of the present invention include at least one 5′-modified phosphate group on a single strand or on at least one 5′-position of a double stranded sequence or sequences. Further modifications are also considered such as internucleoside linkages, conjugate groups, substitute sugars or bases, substitution of one or more nucleosides with nucleoside mimetics and any other modification that can enhance the selected sequence for its intended target. The terms used to describe the conformational geometry of homoduplex nucleic acids are “A Form” for RNA and “B Form” for DNA. The respective conformational geometry for RNA and DNA duplexes was determined from X-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins, Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNA duplexes are more stable and have higher melting temperatures (Tm's) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, NY.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNA biases the sugar toward a C3′ endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry. In addition, the 2′ hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleic acids prefer a C2′ endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.). As used herein, B-form geometry is inclusive of both C2′-endo pucker and O4′-endo pucker. This is consistent with Berger, et. al., Nucleic Acids Research, 1998, 26, 2473-2480, who pointed out that in considering the furanose conformations which give rise to B-form duplexes consideration should also be given to a O4′-endo pucker contribution.

[0150] DNA:RNA hybrid duplexes, however, are usually less stable than pure RNA:RNA duplexes, and depending on their sequence may be either more or less stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533). The stability of the duplex formed between a target RNA and a synthetic sequence is central to therapies such as but not limited to antisense and RNA interference as these mechanisms require the binding of a synthetic oligonucleotide strand to an RNA target strand. In the case of antisense, effective inhibition of the mRNA requires that the antisense DNA have a very high binding affinity with the mRNA. Otherwise the desired interaction between the synthetic oligonucleotide strand and target mRNA strand will occur infrequently, resulting in decreased efficacy.

[0151] One routinely used method of modifying the sugar puckering is the substitution of the sugar at the 2′-position with a substituent group that influences the sugar geometry. The influence on ring conformation is dependant on the nature of the substituent at the 2′-position. A number of different substituents have been studied to determine their sugar puckering effect. For example, 2′-halogens have been studied showing that the 2′-fluoro derivative exhibits the largest population (65%) of the C3′-endo form, and the 2′-iodo exhibits the lowest population (7%). The populations of adenosine (2′-OH) versus deoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, the effect of the 2′-fluoro group of adenosine dimers (21-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is further correlated to the stabilization of the stacked conformation.

[0152] As expected, the relative duplex stability can be enhanced by replacement of 2′-OH groups with 2′-F groups thereby increasing the C3′-endo population. It is assumed that the highly polar nature of the 2′-F bond and the extreme preference for C3′-endo puckering may stabilize the stacked conformation in an A-form duplex. Data from UV hypochromicity, circular dichroism, and ¹H NMR also indicate that the degree of stacking decreases as the electronegativity of the halo substituent decreases. Furthermore, steric bulk at the 2′-position of the sugar moiety is better accommodated in an A-form duplex than a B-form duplex. Thus, a 2′-substituent on the 3′-terminus of a dinucleoside monophosphate is thought to exert a number of effects on the stacking conformation: steric repulsion, furanose puckering preference, electrostatic repulsion, hydrophobic attraction, and hydrogen bonding capabilities. These substituent effects are thought to be determined by the molecular size, electronegativity, and hydrophobicity of the substituent. Melting temperatures of complementary strands is also increased with the 2′-substituted adenosine diphosphates. It is not clear whether the 3′-endo preference of the conformation or the presence of the substituent is responsible for the increased binding. However, greater overlap of adjacent bases (stacking) can be achieved with the 3′-endo conformation.

[0153] One synthetic 2′-modification that imparts increased nuclease resistance and a very high binding affinity to nucleotides is the 2-methoxyethoxy (2′-MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). One of the immediate advantages of the 2′-MOE substitution is the improvement in binding affinity, which is greater than many similar 2′ modifications such as O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-O-methoxyethyl substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926). Relative to DNA, the oligonucleotides having the 2′-MOE modification displayed improved RNA affinity and higher nuclease resistance. Chimeric oligonucleotides having 2′-MOE substituents in the wing nucleosides and an internal region of deoxy-phosphorothioate nucleotides (also termed a gapped oligonucleotide or gapmer) have shown effective reduction in the growth of tumors in animal models at low doses. 2′-MOE substituted oligonucleotides have also shown outstanding promise as antisense agents in several disease states. One such MOE substituted oligonucleotide is presently being investigated in clinical trials for the treatment of CMV retinitis.

[0154] Chemistries Defined

[0155] Unless otherwise defined herein, alkyl means C₁-C₁₂, preferably C₁-C₈ and more preferably C₁-C₆, straight or (where possible) branched chain aliphatic hydrocarbyl.

[0156] Unless otherwise defined herein, heteroalkyl means C₁-C₁₂, preferably C₁-C₈, and more preferably C₁-C₆, straight or (where possible) branched chain aliphatic hydrocarbyl containing at least one, and preferably about 1 to about 3, hetero atoms in the chain, including the terminal portion of the chain. Preferred heteroatoms include N, O and S. Unless otherwise defined herein, cycloalkyl means C₃-C₁₂, preferably C₃-C₈, and more preferably C₃-C₆, aliphatic hydrocarbyl ring.

[0157] Unless otherwise defined herein, alkenyl means C₂-C₁₂, preferably C₂-C₈, and more preferably C₂-C₆ alkenyl, which may be straight or (where possible) branched hydrocarbyl moiety, which contains at least one carbon-carbon double bond.

[0158] Unless otherwise defined herein, alkynyl means C₂-C₁₂, preferably C₂-C₈, and more preferably C₂-C₆ alkynyl, which may be straight or (where possible) branched hydrocarbyl moiety, which contains at least one carbon-carbon triple bond.

[0159] Unless otherwise defined herein, heterocycloalkyl means a ring moiety containing at least three ring members, at least one of which is carbon, and of which 1, 2 or three ring members are other than carbon. Preferably the number of carbon atoms varies from 1 to about 12, preferably 1 to about 6, and the total number of ring members varies from three to about 15, preferably from about 3 to about 8. Preferred ring heteroatoms are N, O and S. Preferred heterocycloalkyl groups include morpholino, thiomorpholino, piperidinyl, piperazinyl, homopiperidinyl, homopiperazinyl, homomorpholino, homothiomorpholino, pyrrolodinyl, tetrahydrooxazolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydroisoxazolyl, tetrahydropyrrazolyl, furanyl, pyranyl, and tetrahydroisothiazolyl.

[0160] Unless otherwise defined herein, aryl means any hydrocarbon ring structure containing at least one aryl ring. Preferred aryl rings have about 6 to about 20 ring carbons. Especially preferred aryl rings include phenyl, napthyl, anthracenyl, and phenanthrenyl.

[0161] Unless otherwise defined herein, hetaryl means a ring moiety containing at least one fully unsaturated ring, the ring consisting of carbon and non-carbon atoms. Preferably the ring system contains about 1 to about 4 rings. Preferably the number of carbon atoms varies from 1 to about 12, preferably 1 to about 6, and the total number of ring members varies from three to about 15, preferably from about 3 to about 8. Preferred ring heteroatoms are N, O and S. Preferred hetaryl moieties include pyrazolyl, thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl, pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl, benzothiophenyl, etc.

[0162] Unless otherwise defined herein, where a moiety is defined as a compound moiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl and alkyl), etc., each of the submoieties is as defined herein.

[0163] Unless otherwise defined herein, an electron withdrawing group is a group, such as the cyano or isocyanato group that draws electronic charge away from the carbon to which it is attached. Other electron withdrawing groups of note include those whose electronegativities exceed that of carbon, for example halogen, nitro, or phenyl substituted in the ortho- or para-position with one or more cyano, isothiocyanato, nitro or halo groups.

[0164] Unless otherwise defined herein, the terms halogen and halo have their ordinary meanings. Preferred halo (halogen) substituents are Cl, Br, and I.

[0165] The aforementioned optional substituents are, unless otherwise herein defined, suitable substituents depending upon desired properties. Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties, NO₂, NH₃ (substituted and unsubstituted), acid moieties (e.g. —CO₂H, —OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties, aryl moieties, etc. In all the preceding formulae, the squiggle (˜) indicates a bond to an oxygen or sulfur of the 5′-phosphate.

[0166] Phosphate protecting groups include those described in U.S. patent Nos. U.S. Pat. No. 5,760,209, U.S. Pat. No. 5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat. No. 6,020,475, U.S. Pat. No. 6,326,478, U.S. Pat. No. 6,169,177, U.S. Pat. No. 6,121,437, U.S. Pat. No. 6,465,628 each of which is expressly incorporated herein by reference in its entirety.

[0167] The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of the routineer. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and 2′-alkoxy or 2′-alkoxyalkoxy derivatives, including 2′-O-methoxyethyl oligonucleotides (Martin, P., Helv. Chim. Acta 1995, 78, 486-504). It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling, Va.) to synthesize fluorescently labeled, biotinylated or other conjugated oligonucleotides.

[0168] The antisense compounds of the present invention include bioequivalent compounds, including pharmaceutically acceptable salts and prodrugs. This is intended to encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of the nucleic acids of the invention and prodrugs of such nucleic acids. A pharmaceutically acceptable salts@ are physiologically and pharmaceutically acceptable salts of the nucleic acids of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci. 1977, 66, 1-19).

[0169] For oligonucleotides, examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; 8 salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

[0170] The oligonucleotides of the invention may additionally or alternatively be prepared to be delivered in a Aprodrug@ form. The term Aprodrug@ indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510.

[0171] For therapeutic or prophylactic treatment, oligonucleotides are administered in accordance with this invention. Oligonucleotide compounds of the invention may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like in addition to the oligonucleotide. Such compositions and formulations are comprehended by the present invention.

[0172] Pharmaceutical compositions comprising the oligonucleotides of the present invention may include penetration enhancers in order to enhance the alimentary delivery of the oligonucleotides. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., fatty acids, bile salts, chelating agents, surfactants and non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, 8, 91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33). One or more penetration enhancers from one or more of these broad categories may be included. Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myristic 35 acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono- and di-glycerides and physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1; El-Hariri et al., J. Pharm. Pharmacol. 1992 44, 651-654).

[0173] The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, NY, 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term “bile salt” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.

[0174] Complex formulations comprising one or more penetration enhancers may be used. For example, bile salts may be used in combination with fatty acids to make complex formulations.

[0175] Chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33; Buur et al., J. Control Rel. 1990, 14, 43-51). Chelating agents have the added advantage of also serving as DNase inhibitors.

[0176] Surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Pharmacol. 1988, 40, 252-257).

[0177] Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol. 1987, 39, 621-626).

[0178] As used herein, “carrier compound” refers to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. In contrast to a carrier compound, a “pharmaceutically acceptable carrier” (excipient) is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The pharmaceutically acceptable carrier may be liquid or solid and is selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.); or. wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are described in U.S. Pat. Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309,404.

[0179] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the invention.

[0180] Regardless of the method by which the oligonucleotides of the invention are introduced into a patient, colloidal dispersion systems may be used as delivery vehicles to enhance the in vivo stability of the oligonucleotides and/or to target the oligonucleotides to a particular organ, tissue or cell type. Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:oligonucleotide complexes of uncharacterized structure. A preferred colloidal dispersion system is a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration (see, generally, Chonn et al., Current Op. Biotech. 1995, 6, 698-708).

[0181] The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, epidermal, and transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

[0182] Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0183] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0184] Compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. In some cases it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities in order to increase the efficacy of a treatment regimen. In the context of the invention, the term “treatment regimen” is meant to encompass therapeutic, palliative and prophylactic modalities. For example, a patient may be treated with conventional chemotherapeutic agents such as those used for tumor and cancer treatment. When used with the compounds of the invention, such chemotherapeutic agents may be used individually, sequentially, or in combination with one or more other such chemotherapeutic agents.

[0185] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in vitro and in in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0186] Thus, in the context of this invention, by “therapeutically effective amount” is meant the amount of the compound which is required to have a therapeutic effect on the treated individual. This amount, which will be apparent to the skilled artisan, will depend upon the age and weight of the individual, the type of disease to be treated, perhaps even the gender of the individual, and other factors which are routinely taken into consideration when designing a drug treatment. A therapeutic effect is assessed in the individual by measuring the effect of the compound on the disease state in the animal.

[0187] The following examples illustrate the present invention and are not intended to limit the same.

EXAMPLES Example 1 Synthesis of Oligonucleotides

[0188] Unmodified oligodeoxynucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. β-cyanoethyldiisopropyl-phosphoramidites are purchased from Applied Biosystems (Foster City, Calif.). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of ³H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step was increased to 68 seconds and was followed by the capping step. Cytosines may be 5-methyl cytosines. (5-methyl deoxycytidine phosphoramidites available from Glen Research, Sterling, Va. or Amersham Pharmacia Biotech, Piscataway, N.J.)

[0189] 2′-methoxy oligonucleotides are synthesized using 2′-methoxy β-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham, Mass.) and the standard cycle for unmodified oligonucleotides, except the wait step after pulse delivery of tetrazole and base is increased to 360 seconds. Other 2′-alkoxy oligonucleotides are synthesized by a modification of this method, using appropriate 2′-modified amidites such as those available from Glen Research, Inc., Sterling, Va.

[0190] 2′-fluoro oligonucleotides are synthesized as described in Kawasaki et al. (J. Med. Chem. 1993, 36, 831-841). Briefly, the protected nucleoside N⁶-benzoyl-2′-deoxy-2′-fluoroadenosine is synthesized utilizing commercially available 9-β-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-α-fluoro atom is introduced by a S_(N)2-displacement of a 2′-β-O-trifyl group. Thus N⁶-benzoyl-9-β-D-arabinofuranosyladenine is selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N⁶-benzoyl groups is accomplished using standard methodologies. Standard methods are also used to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0191] The synthesis of 2′-deoxy-2′-fluoroguanosine is accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-β-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyrylarabinofuranosylguanosine. Deprotection of the TPDS group is followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation is followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies are used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.

[0192] Synthesis of 2′-deoxy-2′-fluorouridine is accomplished by the modification of a known procedure in which 2, 2′-anhydro-1-β-D-arabinofuranosyluracil is treated with 70% hydrogen fluoride-pyridine. Standard procedures are used to obtain the 5¹-DMT and 5′-DMT-3′phosphoramidites.

[0193] 2′-deoxy-2′-fluorocytidine is synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N⁴-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures are used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0194] 2′-(2-methoxyethyl)-modified amidites were synthesized according to Martin, P. (Helv. Chim. Acta 1995, 78, 486-506). For ease of synthesis, the last nucleotide may be a deoxynucleotide. 2′-O—CH₂CH₂OCH₃cytosines may be 5-methyl cytosines.

[0195] Synthesis of 5-Methyl Cytosine Monomers

[0196] 2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]

[0197] 5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60EC at 1 mm Hg for 24 hours) to give a solid which was crushed to a light tan powder (57 g, 85% crude yield). The material was used as is for further reactions.

[0198] 2′-O-Methoxyethyl-5-methyluridine

[0199] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160EC. After heating for 48 hours at 155-160EC, the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product.

[0200] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0201] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH₃CN (200 mL) . The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).

[0202] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-uridine

[0203] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by tlc by first quenching the tlc sample with the addition of MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL) was added and the mixture evaporated at 35EC. The residue was dissolved in CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl₃. The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/Hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%).

[0204] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0205] A first solution was prepared by dissolving 3¹-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L), cooled to −5EC and stirred for 0.5 hours using an overhead stirrer. POCl₃ was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10EC, and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the later solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.

[0206] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0207] A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH₃ gas was added and the vessel heated to 100EC for 2 hours (tlc showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.

[0208] N⁴-Benzoyl-2′-O-methoxyethyl-51-O-dimethoxytrityl-5-methylcytidine

[0209] 2′-O-Methoxyethyl-51-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tlc showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.

[0210] N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite:

[0211] N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (tlc showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH₂Cl₂ (300 mL), and the extracts were combined, dried over MgSO₄ and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAcHexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.

[0212] 5-methyl-2′-deoxycytidine (5-me-C) containing oligonucleotides were synthesized according to published methods (Sanghvi et al., Nucl. Acids Res. 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

[0213] Oligonucleotides having methylene(methylimino) (MMI) backbones were synthesized according to U.S. Pat. No. 5,378,825, which is coassigned to the assignee of the present invention and is incorporated herein in its entirety. For ease of synthesis, various nucleoside dimers containing MMI linkages were synthesized and incorporated into oligonucleotides. Other nitrogen-containing backbones are synthesized according to WO 92/20823 which is also coassigned to the assignee of the present invention and incorporated herein in its entirety.

[0214] Oligonucleotides having amide backbones are synthesized according to De Mesmaeker et al. (Acc. Chem. Res. 1995, 28, 366-374). The amide moiety is readily accessible by simple and well-known synthetic methods and is compatible with the conditions required for solid phase synthesis of oligonucleotides.

[0215] Oligonucleotides with morpholino backbones are synthesized according to U.S. Pat. No. 5,034,506 (Summerton and Weller).

[0216] Peptide-nucleic acid (PNA) oligomers are synthesized according to P. E. Nielsen et al. (Science 1991, 254, 1497-1500). After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55EC for 18 hours, the oligonucleotides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by ³¹P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al. (J. Biol. Chem. 1991, 266, 18162). Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 2 Human TNF-α Oligodeoxynucleotide Sequences

[0217] Antisense oligonucleotides were designed to target human TNF-α. Target sequence data are from the TNFα cDNA sequence published by Nedwin, G. E. et al. (Nucleic Acids Res. 1985, 13, 6361-6373); Genbank accession number X02910, provided herein as SEQ ID NO: 1. Oligodeoxynucleotides were synthesized primarily with phosphorothioate linkages. Oligonucleotide sequences are shown in Table 1. Oligonucleotide 14640 (SEQ ID NO. 2) is a published TNF-α antisense oligodeoxynucleotide targeted to the start site of the TNFα gene (Hartmann, G., et al., Antisense Nucleic Acid Drug Dev., 1996, 6, 291-299). Oligonucleotide 2302 (SEQ ID NO. 41) is an antisense oligodeoxynucleotide targeted to the human intracellular adhesion molecule-1 (ICAM-1) and was used as an unrelated (negative) target control. Oligonucleotide 13664 (SEQ ID NO. 42) is an antisense oligodeoxynucleotide targeted to the Herpes Simplex Virus type 1 and was used as an unrelated target control.

[0218] NeoHK cells, human neonatal foreskin keratinocytes (obtained from Cascade Biologicals, Inc., Portland, Oreg.) were cultured in Keratinocyte medium containing the supplied growth factors (Life Technologies, Rockville, Md.).

[0219] At assay time, the cells were between 70% and 90% confluent. The cells were incubated in the presence of Keratinocyte medium, without the supplied growth factors added, and the oligonucleotide formulated in LIPOFECTIN7 (Life Technologies), a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water. For an initial screen, the oligonucleotide concentration was 300 nM in 9 μg/mL LIPOFECTIN7. Treatment was for four hours. After treatment, the medium was removed and the cells were further incubated in Keratinocyte medium containing the supplied growth factors and 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma, St. Louis, Mo). mRNA was analyzed 2 hours post-induction with PMA. Protein levels were analyzed 12 to 20 hours post-induction.

[0220] Total mRNA was isolated using the RNEASY7 Mini Kit (Qiagen, Valencia, Calif.; similar kits from other manufacturers may also be used), separated on a 1% agarose gel, transferred to HYBOND™-N+ membrane (Amersham Pharmacia Biotech, Piscataway, N.J.), a positively charged nylon membrane, and probed. A TNF-α probe consisted of the 505 bp EcoRI-HindIII fragment from BBG 18 (R&D Systems, Minneapolis, Minn.), a plasmid containing human TNF-α cDNA. A glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe consisted of the 1.06 kb HindIII fragment from pHcGAP (American Type Culture Collection, Manassas, Va.), a plasmid containing human G3PDH cDNA. The restriction fragments were purified from low-melting temperature agarose, as described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, 1989 and labeled with REDIVUE™ ³²P-dCTP (Amersham Pharmacia Biotech, Piscataway, N.J.) and PRIME-A-GENE7 labeling kit (Promega, Madison, Wis.). mRNA was quantitated by a PhosphoImager (Molecular Dynamics, Sunnyvale, Calif.). Secreted TNF-α protein levels were measured using a human TNF-α ELIZA kit (R&D Systems, Minneapolis, Minn. or Genzyme, Cambridge, Mass.). TABLE 1 Nucleotide Sequences of Human TNF-α Phosphorothioate Oligodeoxynucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′ -> 3′) NO: ORDINATES² REGION 14640 CATGCTTTCAGTGCTCAT  2 0796-0813 AUG 14641 TGAGGGAGCGTCTGCTGGCT  3 0615-0634 5′-UTR 14642 GTGCTCATGGTGTCCTTTCC  4 0784-0803 AUG 14643 TAATCACAAGTGCAAACATA  5 3038-3057 3′-UTR 14644 TACCCCGGTCTCCCAAATAA  6 3101-3120 3′-UTR 14810 GTGCTCATGGTGTCCTTTCC  4 0784-0803 AUG 14811 AGCACCGCCTGGAGCCCT  7 0869-0886 coding 14812 GCTGAGGAACAAGCACCGCC  8 0878-0897 coding 14813 AGGCAGAAGAGCGTGGTGGC  9 0925-0944 coding 14814 AAAGTGCAGCAGGCAGAAGA 10 0935-0954 coding 14815 TTAGAGAGAGGTCCCTGG 11 1593-1610 coding 14816 TGACTGCCTGGGCCAGAG 12 1617-1634 junction 14817 GGGTTCGAGAAGATGATC 13 1822-1839 junction 14818 GGGCTACAGGCTTGTCACTC 14 1841-1860 coding 14820 CCCCTCAGCTTGAGGGTTTG 15 2171-2190 junction 14821 CCATTGGCCAGGAGGGCATT 16 2218-2237 coding 14822 ACCACCAGCTGGTTATCTCT 17 2248-2267 coding 14823 CTGGGAGTAGATGAGGTACA 18 2282-2301 coding 14824 CCCTTGAAGAGGACCTGGGA 19 2296-2315 coding 14825 GGTGTGGGTGAGGAGCACAT 20 2336-2355 coding 14826 GTCTGGTAGGAGACGGCGAT 21 2365-2384 coding 14827 GCAGAGAGGAGGTTGACCTT 22 2386-2405 coding 14828 GCTTGGCCTCAGCCCCCTCT 23 2436-2455 coding 14829 CCTCCCAGATAGATGGGCTC 24 2464-2483 coding 14830 CCCTTCTCCAGCTGGAAGAC 25 2485-2504 coding 14831 ATCTCAGCGCTGAGTCGGTC 26 2506-2525 coding 14832 TCGAGATAGTCGGGCCGATT 27 2527-2546 coding 14833 AAGTAGACCTGCCCAGACTC 28 2554-2573 coding 14834 GGATGTTCGTCCTCCTCACA 29 2588-2607 STOP 14835 ACCCTAAGCCCCCAATTCTC 30 2689-2708 3′-UTR 14836 CCACACATTCCTGAATCCCA 31 2758-2777 3′-UTR 14837 AGGCCCCAGTGAGTTCTGGA 32 2825-2844 3′-UTR 14838 GTCTCCAGATTCCAGATGTC 33 2860-2879 3′-UTR 14839 CTCAAGTCCTGCAGCATTCT 34 2902-2921 3′-UTR 14840 TGGGTCCCCCAGGATACCCC 35 3115-3134 3′-UTR 14841 ACGGAAAACATGTCTGAGCC 36 3151-3170 3′-UTR 14842 CTCCGTTTTCACGGAAAACA 37 3161-3180 3′-UTR 14843 GCCTATTGTTCAGCTCCGTT 38 3174-3193 3′-UTR 14844 GGTCACCAAATCAGCATTGT 39 3272-3292 3′-UTR 14845 GAGGCTCAGCAATGAGTGAC 40 3297-3316 3′-UTR  2302 GCCCAAGCTGGCATCCGTCA 41 target control 13664 GCCGAGGTCCATGTCGTACGC 42 target control

[0221] Results are shown in Table 2. Oligonucleotides 14828 (SEQ ID NO. 23), 14829 (SEQ ID NO. 24), 14832 (SEQ ID NO. 27), 14833 (SEQ ID NO. 28), 14834 (SEQ ID NO. 29), 14835 (SEQ ID NO. 30), 14836 (SEQ ID NO. 31), 14839 (SEQ ID NO. 34), 14840 (SEQ ID NO. 35), and 14844 (SEQ ID NO. 39) inhibited TNFα expression by approximately 50% or more. Oligonucleotides 14828 (SEQ ID NO. 23), 14834 (SEQ ID NO. 29), and 14840 (SEQ ID NO. 35) gave better than 70% inhibition. TABLE 2 Inhibition of Human TNF-α mRNA Expression by Phosphorothioate Oligodeoxynucleotides ISIS SEQ ID GENE TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION basal — — 16% — induced — — 100%  0% 13664 42 control 140% — 14640  2 AUG 61% 39% 14641  3 5′-UTR 95%  5% 14642  4 AUG 131% — 14810  4 AUG 111% — 14815 11 coding 85% 15% 14816 12 junction 106% — 14817 13 junction 97%  3% 14818 14 coding 64% 36% 14820 15 junction 111% — 14821 16 coding 91%  9% 14822 17 coding 57% 43% 14827 22 coding 67% 33% 14828 23 coding 27% 73% 14829 24 coding 33% 67% 14830 25 coding 71% 29% 14831 26 coding 62% 38% 14832 27 coding 40% 60% 14833 28 coding 43% 57% 14834 29 STOP 26% 74% 14835 30 3′-UTR 32% 68% 14836 31 3′-UTR 40% 60% 14837 32 3′-UTR 106% — 14838 33 3′-UTR 70% 30% 14839 34 5′-UTR 49% 51% 14840 35 3′-UTR 28% 72% 14841 36 3′-UTR 60% 40% 14842 37 3′-UTR 164% — 14843 38 3′-UTR 67% 33% 14844 39 3′-UTR 46% 54% 14845 40 3′-UTR 65% 35%

Example 3 Dose Response of Antisense Phosphorothioate Oligodeoxynucleotide Effects on Human TNF-α mRNA Levels in NeoHK Cells

[0222] Four of the more active oligonucleotides from the initial screen were chosen for dose response assays. These include oligonucleotides 14828 (SEQ ID NO. 23), 14833 (SEQ ID NO. 28), 14834 (SEQ ID NO. 29) and 14839 (SEQ ID NO. 34). NeoHK cells were grown, treated and processed as described in Example 2. LIPOFECTIN7 was added at a ratio of 3 μg/mL per 100 nM of oligonucleotide. The control included LIPOFECTIN7 at a concentration of 9 μg/mL. The effect of the TNFα antisense oligonucleotides was normalized to the non-specific target control. Results are shown in Table 3. Each oligonucleotide showed a dose response effect with maximal inhibition greater than 70%. Oligonucleotides 14828 (SEQ ID NO. 23) had an IC₅₀ of approximately 185 nM. Oligonucleotides 14833 (SEQ ID NO. 28) had an IC₅₀ of approximately 150 nM. Oligonucleotides 14834 (SEQ ID NO. 29) and 14839 (SEQ ID NO. 34) had an IC₅₀ of approximately 140 nM. TABLE 3 Dose Response of NeoHK Cells to TNF-α Antisense Phosphorothioate Oligodeoxynucleotides (ASOs) ASO Gene % mRNA % mRNA ISIS # SEQ ID NO: Target Dose Expression Inhibition  2302 41 control  25 nM 100% — ″ ″ ″  50 nM 100% — ″ ″ ″ 100 nM 100% — ″ ″ ″ 200 nM 100% — ″ ″ ″ 300 nM 100% — 14828 23 coding  25 nM 122% — ″ ″ ″  50 nM 97%  3% ″ ″ ″ 100 nM 96%  4% ″ ″ ″ 200 nM 40% 60% ″ ″ ″ 300 nM 22% 78% 14833 28 coding  25 nM 89% 11% ″ ″ ″  50 nM 8% 22% ″ ″ ″ 100 nM 64% 36% ″ ″ ″ 200 nM 36% 64% ″ ″ ″ 300 nM 25% 75% 14834 29 STOP  25 nM 94%  6% ″ ″ ″  50 nM 69% 31% ″ ″ ″ 100 nM 65% 35% ″ ″ ″ 200 nM 26% 74% ″ ″ ″ 300 nM 11% 89% 14839 34 3′-UTR  25 nM 140% — ″ ″ ″  50 nM 112% — ″ ″ ″ 100 nM 65% 35% ″ ″ ″ 200 nM 29% 71% ″ ″ ″ 300 nM 22% 78%

Example 4 Design and Testing of Chimeric (Deoxy Gapped) 2′-O-methoxyethyl TNF-α Antisense Oligonucleotides on TNF-α Levels in NeoHK Cells

[0223] Oligonucleotides having SEQ ID NO: 28 and SEQ ID NO: 29 were synthesized as uniformly phosphorothioate or mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. The sequences and the oligonucleotide chemistries are shown in Table 4. All 2′-MOE cytosines were 5-methyl-cytosines.

[0224] Dose response experiments, as discussed in Example 3, were performed using these chimeric oligonucleotides. The effect of the TNF-α antisense oligonucleotides was normalized to the non-specific target control. Results are shown in Table 5. The activities of the chimeric oligonucleotides tested were comparable to the parent phosphorothioate oligonucleotide. TABLE 4 Nucleotide Sequences of TNF-α Chimeric (deoxy gapped) 2′-O-methoxyethyl Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5′ -> 3′)¹ NO: CO-ORDINATES² REGION 14833 AsAsGsTsAsGsAsCsCsTsGsCsCsCsAsGsAsCsTsC 28 2554-2573 coding 16467 AoAoGoToAsGsAsCsCsTsGsCsCsCsAsGoAoCoToC 28 2554-2573 coding 16468 AsAsGsTsAsGsAsCsCsTsGsCsCsCsAsGsAsCsTsC 28 2554-2573 coding 16469 AsAsGsTsAsGsAsCsCsTsGsCsCsCsAsGsAsCsTsC 28 2554-2573 coding 16470 AsAsGsTsAsGsAsCsCsTsGsCsCsCsAsGsAsCsTsC 28 2554-2573 coding 16471 AsAsGsTsAsGsAsCsCsTsGsCsCsCsAsGsAsCsTsC 28 2554-2573 coding 14834 GsGsAsTsGsTsTsCsGsTsCsCsTsCsCsTsCsAsCsA 29 2588-2607 STOP 16472 GoGoAoToGsTsTsCsGsTsCsCsTsCsCsToCoAoCoA 29 2588-2607 STOP 16473 GsGsAsTsGsTsTsCsGsTsCsCsTsCsCsTsCsAsCsA 29 2588-2607 STOP 16474 GsGsAsTsGsTsTsCsGsTsCsCsTsCsCsTsCsAsCsA 29 2588-2607 STOP 16475 GsGsAsTsGsTsTsCsGsTsCsCsTsCsCsTsCsAsCsA 29 2588-2607 STOP 16476 GsGsAsTsGsTsTsCsGsTsCsCsTsCsCsTsCsAsCsA 29 2588-2607 STOP

[0225] TABLE 5 Dose Response of NeoHK Cells to TNF-α Chimeric (deoxy gapped) 2′-O-methoxyethyl Antisense Oligonucleotides ASO Gene % mRNA % mRNA ISIS # SEQ ID NO: Target Dose Expression Inhibition 13664 42 Control  50 nM 100% — ″ ″ ″ 100 nM 100% — ″ ″ ″ 200 nM 100% — ″ ″ ″ 300 nM 100% — 14833 28 Coding  50 nM 69% 31% ″ ″ ″ 100 nM 64% 36% ″ ″ ″ 200 nM 56% 44% ″ ″ ″ 300 nM 36% 64% 16468 28 Coding  50 nM 66% 34% ″ ″ ″ 100 nM 53% 47% ″ ″ ″ 200 nM 34% 66% ″ ″ ″ 300 nM 25% 75% 16471 28 Coding  50 nM 77% 23% ″ ″ ″ 100 nM 56% 44% ″ ″ ″ 200 nM 53% 47% ″ ″ ″ 300 nM 31% 69% 14834 29 STOP  50 nM 74% 26% ″ ″ ″ 100 nM 53% 47% ″ ″ ″ 200 nM 24% 76% ″ ″ ″ 300 nM 11% 89% 16473 29 STOP  50 nM 71% 29% ″ ″ ″ 100 nM 51% 49% ″ ″ ″ 200 nM 28% 72% ″ ″ ″ 300 nM 23% 77% 16476 29 STOP  50 nM 74% 26% ″ ″ ″ 100 nM 58% 42% ″ ″ ″ 200 nM 32% 68% ″ ″ ″ 300 nM 31% 69%

Example 5 Design and Testing of Chimeric Phosphorothioate/MMI TNF-α Antisense Oligodeoxynucleotides on TNF-α Levels in NeoHK Cells

[0226] Oligonucleotides having SEQ ID NO. 29 were synthesized as mixed phosphorothioate/methylene(methylimino) (MMI) chimeric oligodeoxynucleotides. The sequences and the oligonucleotide chemistries are shown in Table 6. Oligonucleotide 13393 (SEQ ID NO. 49) is an antisense oligonucleotide targeted to the human intracellular adhesion molecule-1 (ICAM-1) and was used as an unrelated target control. All cytosines were 5-methyl-cytosines.

[0227] Dose response experiments were performed using these chimeric oligonucleotides, as discussed in Example 3 except quantitation of TNFα mRNA levels was determined by real-time PER (RT-PER) using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PER) products in real-time. As opposed to standard PER, in which amplification products are quantitated after the PER is completed, products in RT-PER are quantitated as they accumulate. This is accomplished by including in the PER reaction an oligonucleotide probe that anneals specifically between the forward and reverse PER primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE or FAM, PE-Applied Biosystems, Foster City, CA) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PER amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular (six-second) intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0228] RT-PER reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PER reactions were carried out by adding 25 μl PER cocktail (1× TAQMAN7 buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 U RNAse inhibitor, 1.25 units AMPLITAQ GOLD7, and 12.5 U MuLV reverse transcriptase) to 96 well plates containing 25 μl poly(A) mRNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD7, 40 cycles of a two-step PER protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0229] For TNF-α the PER primers were: Forward: 5′-CAGGCGGTGCTTGTTCCT-3′ SEQ ID NO. 43 Reverse: 5′-GCCAGAGGGCTGATTAGAGAGA-3′ SEQ ID NO. 44 and the PER probe was: FAM-CTTCTCCTTCCTGATCGTGGCAGGC-TAMRA (SEQ ID NO. 45) where FAM or JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

[0230] For GAPDH the PER primers were: Forward primer: 5′-GAAGGTGAAGGTCGGAGTC-3′ SEQ ID NO. 46 Reverse primer: 5′-GAAGATGGTGATGGGATTTC-3′ SEQ ID NO. 47 and the PER probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO. 48) where FAM or JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

[0231] Results are shown in Table 7. The oligonucleotide containing MMI linkages was more effective in reducing TNFα mRNA levels than the uniformly phosphorothioate oligonucleotide. The IC₅₀ value was reduced from approximately 75 nM, for oligonucleotide 14834 (SEQ ID NO: 29), to approximately 30 nM for oligonucleotide 16922 (SEQ ID NO: 29).

[0232] Dose response experiments were also performed measuring the effect on TNF-α protein levels. Protein levels were measured as described in Example 2. Results are shown in Table 8. The oligonucleotide containing four MMI linkages on each end was more effective in reducing protein levels than the uniformly phosphorothioate oligonucleotide. The IC₅₀ value was reduced from approximately 90 nM, for oligonucleotide 14834 (SEQ ID NO: 29), to approximately 45 nM for oligonucleotide 16922 (SEQ ID NO: 29). TABLE 6 Nucleotide Sequences of Human TNF-α Chimeric Phosphorothioate/MMI Oligodeoxynucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES² REGION 14834 GsGsAsTsGsTsTsCsGsTsCsCsTsCsCsTsCsAsCsA 29 2588-2607 STOP 16922 GmGmAmTmGsTsTsCsGsTsCsCsTsCsCsTmCmAmCmA 29 2588-2607 STOP 16923 GmGmAmTmGmTmTsCsGsTsCsCsTsCmCmTmCmAmCmA 29 2588-2607 STOP 13393 TsCsTsGsAsGsTsAsGsCsAsGsAsGsGsAsGsCsTsC 49 target control

[0233] TABLE 7 Dose Response of Chimeric Phosphorothioate/MMI TNF-α Antisense Oligodeoxynucleotides on TNF-α mRNA Levels in PMA-Induced NeoHK Cells ASO Gene % mRNA % mRNA ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — —  100% — 13393 49 control  25 nM 87.3% 12.7% ″ ″ ″  50 nM 98.5%  1.5% ″ ″ ″ 100 nM 133.1%  — ″ ″ ″ 200 nM 139.6%  — 14834 29 STOP  25 nM 98.7%  1.3% ″ ″ ″  50 nM 70.8% 29.2% ″ ″ ″ 100 nM 36.0% 64.0% ″ ″ ″ 200 nM 38.2% 61.8% 16922 29 STOP  25 nM 58.9% 41.1% ″ ″ ″  50 nM 28.2% 71.8% ″ ″ ″ 100 nM 22.2% 77.8% ″ ″ ″ 200 nM 18.9% 81.1%

[0234] TABLE 8 Dose Response of Chimeric Phosphorothioate/MMI TNF-α Antisense Oligodeoxynucleotides on TNF-α Protein Levels in PMA-Induced NeoHK Cells ASO Gene % protein % protein ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — — 100.0% — 13393 49 control  25 nM 117.0% — ″ ″ ″  50 nM 86.6% 13.4% ″ ″ ″ 100 nM 98.7%  1.3% ″ ″ ″ 200 nM 78.0% 22.0% 14834 29 STOP  25 nM 84.8% 15.2% ″ ″ ″  50 nM 76.9% 23.1% ″ ″ ″ 100 nM 44.5% 55.5% ″ ″ ″ 200 nM 18.7% 81.3% 16922 29 STOP  25 nM 67.1% 32.9% ″ ″ ″  50 nM 48.6% 51.4% ″ ″ ″ 100 nM 20.0% 80.0% ″ ″ ″ 200 nM 7.9% 92.1% 16923 29 STOP  25 nM 79.9% 20.1% ″ ″ ″  50 nM 69.9% 30.1% ″ ″ ″ 100 nM 56.0% 44.0% ″ ″ ″ 200 nM 44.5% 55.5%

Example 6 Additional Human TNFα Antisense Oligonucleotide Sequences

[0235] A second screening of human TNF-α antisense oligonucleotides was performed. Oligonucleotides were designed specifically against specific regions of the TNF-α gene. A series of oligonucleotides was designed to target introns 1 and 3, and exon 4. Sequences targeting introns 1 or 3 were synthesized as uniformly phosphorothioate oligodeoxynucleotides or mixed phosphorothioate/phosphodiester chimeric backbone oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. Sequences targeting exon 4 were synthesized as mixed phosphorothioate/phosphodiester chimeric backbone oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. The sequences of the chimeric oligonucleotides are shown in Table 9. Sequences of the uniformly phosphorothioate oligodeoxynucleotides are shown in Table 11. These oligonucleotides were screened at 50 nM and 200 nM for their ability to inhibit TNFα protein secretion, essentially as described in Example 2. Results for the chimeric backbone oligonucleotides are shown in Table 10; results for the uniformly phosphorothioate oligodeoxynucleotides are shown in Table 12.

[0236] For the chimeric backbone oligonucleotides targeting introns 1 or 3, oligonucleotide 21688 (SED ID NO. 69) gave 60% inhibition or greater. For chimeric backbone oligonucleotides targeting exon 4, two-thirds of the oligonucleotides gave nearly 60% inhibition or greater (SEQ ID NOs. 88, 90, 91, 92, 93, 94, 97, and 98). See Table 10. For the uniformly phosphorothioate oligodeoxynucleotides, five of nine oligonucleotides targeting intron 3 were effective in reducing TNF-α expression by nearly 60% or greater (SEQ ID NOs. 79, 80, 81, 82, and 84). See Table 12.

[0237] Oligonucleotides having SEQ ID NO. 91 and SEQ ID NO. 98 were synthesized as a uniformly phosphorothioate oligodeoxynucleotides or mixed phosphorothioate/phosphodiester chimeric backbone oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. The sequences and the oligonucleotide chemistries are shown in Table 13. All 2′-MOE cytosines and 2′-deoxy cytosines were 5-methyl-cytosines.

[0238] Dose response experiments, as discussed in Example 3, were performed using these oligonucleotides. Included in this experiment were two oligonucleotides targeting intron 1 and two oligonucleotides targeting intron 3. Results are shown in Tables 14 and 15. The oligonucleotides targeting exon 4 with variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides and/or uniformly Ophosphorothioate or mixed phosphorothioate/phosphodiester were, in general, comparable to the parent compound.

[0239] Oligonucleotides targeting introns 1 or 3 having SEQ ID NOs 66, 69 and 80 were effective in reducing TNF-α mRNA levels by greater than 80% and showed a dose response effect with an IC₅₀ approximately 110 nM. See Tables 14 and 15. TABLE 9 Nucleotide Sequences of TNF-α Chimeric Backbone (deoxy gapped) 2′-O-methoxyethyl Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES² REGION 21669 ToGoCoGoTsCsTsCsTsCsAsTsTsTsCsCoCoCoToT 50 1019-1038 intron 1 21670 ToCoCoCoAsTsCsTsCsTsCsTsCsCsCsToCoToCoT 52 1039-1058 intron 1 21671 CoAoGoCoGsCsAsCsAsTsCsTsTsTsCsAoCoCoCoA 52 1059-1078 intron 1 21672 ToCoToCoTsCsTsCsAsTsCsCsCsTsCsCoCoToAoT 53 1079-1098 intron 1 21673 CoGoToCoTsTsTsCsTsCsCsAsTsGsTsToToToToT 54 1099-1118 intron 1 21674 CoAoCoAoTsCsTsCsTsTsTsCsTsGsCsAoToCoCoC 55 1119-1138 intron 1 21675 CoToCoToCsTsTsCsCsCsCsAsTsCsTsCoToToGoC 56 1139-1158 intron 1 21676 GoToCoToCsTsCsCsAsTsCsTsTsTsCsCoToToCoT 57 1159-1178 intron 1 21677 ToToCoCoAsTsGsTsGsCsCsAsGsAsCsAoToCoCoT 58 1179-1198 intron 1 21678 AoToAoCoAsCsAsCsTsTsAsGsTsGsAsGoCoAoCoC 59 1199-1218 intron 1 21679 ToToCoAoTsTsCsAsTsTsCsAsTsTsCsAoCoToCoC 60 1219-1238 intron 1 21680 ToAoToAoTsCsTsGsCsTsTsGsTsTsCsAoToToCoA 61 1239-1258 intron 1 21681 CoToGoToCsTsCsCsAsTsAsTsCsTsTsAoToToToA 62 1259-1278 intron 1 21682 ToCoToCoTsTsCsTsCsAsCsAsCsCsCsCoAoCoAoT 63 1279-1298 intron 1 21683 CoAoCoToTsGsTsTsTsCsTsTsCsCsCsCoCoAoToC 64 1299-1318 intron 1 21684 CoToCoAoCsCsAsTsCsTsTsTsAsTsTsCoAoToAoT 65 1319-1338 intron 1 21685 AoToAoToTsTsCsCsCsGsCsTsCsTsTsToCoToGoT 66 1339-1358 intron 1 21686 CoAoToCoTsCsTsCsTsCsCsTsTsAsGsCoToGoToC 67 1359-1378 intron 1 21687 ToCoToToCsTsCsTsCsCsTsTsAsTsCsToCoCoCoC 68 1379-1398 intron 1 21688 GoToGoToGsCsCsAsGsAsCsAsCsCsCsToAoToCoT 69 1399-1418 intron 1 21689 ToCoToToTsCsCsCsTsGsAsGsTsGsTsCoToToCoT 70 1419-1438 intron 1 21690 AoCoCoToTsCsCsAsGsCsAsTsTsCsAsAoCoAoGoC 71 1439-1458 intron 1 21691 CoToCoCoAsTsTsCsAsTsCsTsGsTsGsToAoToToC 72 1459-1478 intron 1 21692 ToGoAoGoGsTsGsTsCsTsGsGsTsTsTsToCoToCoT 73 1479-1498 intron 1 21693 AoCoAoCoAsTsCsCsTsCsAsGsAsGsCsToCoToToA 74 1871-1890 intron 3 21694 CoToAoGoCsCsCsTsCsCsAsAsGsTsTsCoCoAoAoG 75 1891-1910 intron 3 21695 CoGoGoGoCsTsTsCsAsAsTsCsCsCsCsAoAoAoToC 76 1911-1930 intron 3 21696 AoAoGoToTsCsTsGsCsCsTsAsCsCsAsToCoAoGoC 77 1931-1950 intron 3 21697 GoToCoCoTsTsCsTsCsAsCsAsTsTsGsToCoToCoC 78 1951-1970 intron 3 21698 CoCoToToCsCsCsTsTsGsAsGsCsTsCsAoGoCoGoA 79 1971-1990 intron 3 21699 GoGoCoCoTsGsTsGsCsTsGsTsTsCsCsToCoCoAoC 80 1991-2010 intron 3 21700 CoGoToToCsTsGsAsGsTsAsTsCsCsCsAoCoToAoA 81 2011-2030 intron 3 21701 CoAoCoAoTsCsCsCsAsCsCsTsGsGsCsCoAoToGoA 82 2031-2050 intron 3 21702 GoToCoCoTsCsTsCsTsGsTsCsTsGsTsCoAoToCoC 83 2051-2070 intron 3 21703 CoCoAoCoCsCsCsAsCsAsTsCsCsGsGsToToCoCoT 84 2071-2090 intron 3 21704 ToCoCoToGsGsCsCsCsTsCsGsAsGsCsToCoToGoC 85 2091-2110 intron 3 21705 AoToGoToCsGsGsTsTsCsAsCsTsCsTsCoCoAoCoA 86 2111-2130 intron 3 21706 AoGoAoGoGsAsGsAsGsTsCsAsGsTsGsToGoGoCoC 87 2131-2150 intron 3 21722 GoAoToCoCsCsAsAsAsGsTsAsGsAsCsCoToGoCoC 88 2561-2580 exon 4 21723 CoAoGoAoCsTsCsGsGsCsAsAsAsGsTsCoGoAoGoA 89 2541-2560 exon 4 21724 ToAoGoToCsGsGsGsCsCsGsAsTsTsGsAoToCoToC 90 2521-2540 exon 4 21725 AoGoCoGoCsTsGsAsGsTsCsGsGsTsCsAoCoCoCoT 91 2501-2520 exon 4 21726 ToCoToCoCsAsGsCsTsGsGsAsAsGsAsCoCoCoCoT 92 2481-2500 exon 4 21727 CoCoCoAoGsAsTsAsGsAsTsGsGsGsCsToCoAoToA 93 2461-2480 exon 4 21728 CoCoAoGoGsGsCsTsTsGsGsCsCsTsCsAoGoCoCoC 94 2441-2460 exon 4 21729 CoCoToCoTsGsGsGsGsTsCsTsCsCsCsToCoToGoG 95 2421-2440 exon 4 21730 CoAoGoGoGsGsCsTsCsTsTsGsAsTsGsGoCoAoGoA 96 2401-2420 exon 4 21731 GoAoGoGoAsGsGsTsTsGsAsCsCsTsTsGoGoToCoT 97 2381-2400 exon 4 21732 GoGoToAoGsGsAsGsAsCsGsGsCsGsAsToGoCoGoG 98 2361-2380 exon 4 21733 CoToGoAoTsGsGsTsGsTsGsGsGsTsGsAoGoGoAoG 99 2341-2360 exon 4

[0240] TABLE 10 Dose Response of PMA-Induced neoHK Cells to Chimeric Backbone (deoxy gapped) 2′-O-methoxyethyl TNF-α Antisense Oligonucleotides ASO Gene % protein % protein ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — — 100% — 14834 29 STOP  50 nM 76% 24% ″ ″ ″ 200 nM 16% 84% 21669 50 intron 1  50 nM 134% — ″ ″ ″ 200 nM 114% — 21670 51 intron 1  50 nM 122% — ″ ″ ″ 200 nM 101% — 21671 52 intron 1  50 nM 90% 10% ″ ″ ″ 200 nM 58% 42% 21672 53 intron 1  50 nM 122% — ″ ″ ″ 200 nM 131% — 21673 54 intron 1  50 nM 102% — ″ ″ ″ 200 nM 110% — 21674 55 intron 1  50 nM 111% — ″ ″ ″ 200 nM 96%  4% 21675 56 intron 1  50 nM 114% — ″ ″ ″ 200 nM 99%  1% 21676 57 intron 1  50 nM 107% — ″ ″ ″ 200 nM 96%  4% 21677 58 intron 1  50 nM 86% 14% ″ ″ ″ 200 nM 95%  5% 21678 59 intron 1  50 nM 106% — ″ ″ ″ 200 nM 107% — 21679 60 intron 1  50 nM 75% 25% ″ ″ ″ 200 nM 73% 27% 21680 61 intron 1  50 nM 76% 24% ″ ″ ″ 200 nM 80% 20% 21681 62 intron 1  50 nM 79% 21% ″ ″ ″ 200 nM 82% 18% 21682 63 intron 1  50 nM 102% — ″ ″ ″ 200 nM 88% 12% 21683 64 intron 1  50 nM 80% 20% ″ ″ ″ 200 nM 66% 34% 21684 65 intron 1  50 nM 91%  9% ″ ″ ″ 200 nM 69% 31% 21685 66 intron 1  50 nM 98%  2% ″ ″ ″ 200 nM 90% 10% 21686 67 intron 1  50 nM 97%  3% ″ ″ ″ 200 nM 72% 28% 21687 68 intron 1  50 nM 103% — ″ ″ ″ 200 nM 64% 36% 21688 69 intron 1  50 nM 87% 13% ″ ″ ″ 200 nM 40% 60% 21689 70 intron 1  50 nM 78% 22% ″ ″ ″ 200 nM 74% 26% 21690 71 intron 1  50 nM 84% 16% ″ ″ ″ 200 nM 80% 20% 21691 72 intron 1  50 nM 86% 14% ″ ″ ″ 200 nM 75% 25% 21692 73 intron 1  50 nM 85% 15% ″ ″ ″ 200 nM 61% 39% 21693 74 intron 3  50 nM 81% 19% ″ ″ ″ 200 nM 83% 17% 21694 75 intron 3  50 nM 99%  1% ″ ″ ″ 200 nM 56% 44% 21695 76 intron 3  50 nM 87% 13% ″ ″ ″ 200 nM 84% 16% 21696 77 intron 3  50 nM 103% — ″ ″ ″ 200 nM 86% 14% 21697 78 intron 3  50 nM 99%  1% ″ ″ ″ 200 nM 52% 48% 21698 79 intron 3  50 nM 96%  4% ″ ″ ″ 200 nM 47% 53% 21699 80 intron 3  50 nM 73% 27% ″ ″ ″ 200 nM 84% 16% 21700 81 intron 3  50 nM 80% 20% ″ ″ ″ 200 nM 53% 47% 21701 82 intron 3  50 nM 94%  6% ″ ″ ″ 200 nM 56% 44% 21702 83 intron 3  50 nM 86% 14% ″ ″ ″ 200 nM 97%  3% 21703 84 intron 3  50 nM 88% 12% ″ ″ ″ 200 nM 74% 26% 21704 85 intron 3  50 nM 69% 31% ″ ″ ″ 200 nM 65% 35% 21705 86 intron 3  50 nM 92%  8% ″ ″ ″ 200 nM 77% 23% 21706 87 intron 3  50 nM 95%  5% ″ ″ ″ 200 nM 82% 18% 21722 88 exon 4  50 nM 81% 19% ″ ″ ″ 200 nM 41% 59% 21723 89 exon 4  50 nM 87% 13% ″ ″ ″ 200 nM 74% 26% 21724 90 exon 4  50 nM 68% 32% ″ ″ ″ 200 nM 33% 67% 21725 91 exon 4  50 nM 55% 45% ″ ″ ″ 200 nM 30% 70% 21726 92 exon 4  50 nM 72% 28% ″ ″ ″ 200 nM 40% 60% 21727 93 exon 4  50 nM 67% 33% ″ ″ ″ 200 nM 40% 60% 21728 94 exon 4  50 nM 62% 38% ″ ″ ″ 200 nM 41% 59% 21729 95 exon 4  50 nM 78% 22% ″ ″ ″ 200 nM 53% 47% 21730 96 exon 4  50 nM 68% 32% ″ ″ ″ 200 nM 48% 52% 21731 97 exon 4  50 nM 77% 23% ″ ″ ″ 200 nM 41% 59% 21732 98 exon 4  50 nM 62% 38% ″ ″ ″ 200 nM 28% 72% 21733 99 exon 4  50 nM 92%  8% ″ ″ ″ 200 nM 74% 26%

[0241] TABLE 11 Nucleotide Sequences of Additional Human TNF-α Phosphorothioate Oligodeoxynucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES² REGION 21804 TGCGTCTCTCATTTCCCCTT 50 1019-1038 intron 1 21805 TCCCATCTCTCTCCCTCTCT 51 1039-1058 intron 1 21806 CAGCGGACATCTTTCACCCA 52 1059-1078 intron 1 21807 TCTCTCTCATCCCTCCCTAT 53 1079-1098 intron 1 21808 CGTCTTTCTCCATGTTTTTT 54 1099-1118 intron 1 21809 CACATCTCTTTCTGCATCCC 55 1119-1138 intron 1 21810 CTCTCTTCCCCATCTCTTGC 56 1139-1158 intron 1 21811 GTCTCTCCATCTTTCCTTCT 57 1159-1178 intron 1 21812 TTCCATGTGCCAGACATCCT 58 1179-1198 intron 1 21813 ATACACACTTAGTGAGCACC 59 1199-1218 intron 1 21814 TTCATTCATTCATTCACTCC 60 1219-1238 intron 1 21815 TATATCTGCTTGTTCATTCA 61 1239-1258 intron 1 21816 CTGTCTCCATATCTTATTTA 62 1259-1278 intron 1 21817 TCTCTTCTCACACCCCACAT 63 1279-1298 intron 1 21818 CACTTGTTTCTTCCCCCATC 64 1299-1318 intron 1 21819 CTCACCATCTTTATTCATAT 65 1319-1338 intron 1 21820 ATATTTCCCGCTCTTTCTGT 66 1339-1358 intron 1 21821 CATCTCTCTCCTTAGCTGTC 67 1359-1378 intron 1 21822 TCTTCTCTCCTTATCTCCCC 68 1379-1398 intron 1 21823 GTGTGCCAGACACCCTATCT 69 1399-1418 intron 1 21824 TCTTTCCCTGAGTGTCTTCT 70 1419-1438 intron 1 21825 ACCTTCCAGCATTCAACAGC 71 1439-1458 intron 1 21826 CTCCATTCATCTGTGTATTC 72 1459-1478 intron 1 21827 TGAGGTGTCTGGTTTTCTCT 73 1479-1498 intron 1 21828 ACACATCCTCAGAGCTCTTA 74 1871-1890 intron 3 21829 CTAGCCCTCCAAGTTCCAAG 75 1891-1910 intron 3 21830 CGGGCTTCAATCCCCAAATC 76 1911-1930 intron 3 21831 AAGTTCTGCCTACCATCAGC 77 1931-1950 intron 3 21832 GTCCTTCTCACATTGTCTCC 78 1951-1970 intron 3 21833 CCTTCCCTTGAGCTCAGCGA 79 1971-1990 intron 3 21834 GGCCTGTGCTGTTCCTCCAC 80 1991-2010 intron 3 21835 CGTTCTGAGTATCCCACTAA 81 2011-2030 intron 3 21836 CACATCCCACCTGGCCATGA 82 2031-2050 intron 3 21837 GTCCTCTCTGTCTGTCATCC 83 2051-2070 intron 3 21838 CCACCCCACATCCGGTTCCT 84 2071-2090 intron 3 21839 TCCTGGCCCTCGAGCTCTGC 85 2091-2110 intron 3 21840 ATGTCGGTTCACTCTCCACA 86 2111-2130 intron 3 21841 AGAGGAGAGTCAGTGTGGCC 87 2131-2150 intron 3

[0242] TABLE 12 Dose Response of PMA-Induced neoHK Cells to TNF-α Antisense Phosphorothioate Oligodeoxynucleotides ASO Gene % protein % protein ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — — 100% — 14834 29 STOP  50 nM 80% 20% ″ ″ ″ 200 nM 13% 87% 21812 58 intron 1  50 nM 110% — ″ ″ ″ 200 nM 193% — 21833 79 intron 3  50 nM 88% 12% ″ ″ ″ 200 nM 8% 92% 21834 80 intron 3  50 nM 70% 30% ″ ″ ″ 200 nM 18% 82% 21835 81 intron 3  50 nM 106% — ″ ″ ″ 200 nM 42% 58% 21836 82 intron 3  50 nM 71% 29% ″ ″ ″ 200 nM 12% 88% 21837 83 intron 3  50 nM 129% — ″ ″ ″ 200 nM 74% 26% 21838 84 intron 3  50 nM 85% 15% ″ ″ ″ 200 nM 41% 59% 21839 85 intron 3  50 nM 118% — ″ ″ ″ 200 nM 58% 42% 21840 86 intron 3  50 nM 120% — ″ ″ ″ 200 nM 96%  4% 21841 87 intron 3  50 nM 117% — ″ ″ ″ 200 nM 78% 22%

[0243] TABLE 13 Nucleotide Sequences of TNF-α Chimeric (deoxy gapped) 2′-O-Methoxyethyl Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES² REGION 21725 AoGoCoGoCsTsGsAsGsTsCsGsGsTsCsAoCoCoCoT 91 2501-2520 exon 4 25655 AsGsCsGsCsTsGsAsGsTsCsGsGsTsCsAsCsCsCsT ″ ″ ″ 25656 AsGsCsGsCsTsGsAsGsTsCsGsGsTsCsAsCsCsCsT ″ ″ ″ 25660 AoGoCoGsCsTsGsAsGsTsCsGsGsTsCsAsCoCoCoT ″ ″ ″ 21732 GoGoToAoGsGsAsGsAsCsGsGsCsGsAsToGoCoGoG 98 2361-2380 exon 4 25657 GsGsTsAsGsGsAsGsAsCsGsGsCsGsAsTsGsCsGsG ″ ″ ″ 25658 GsGsTsAsGsGsAsGsAsCsGsGsCsGsAsTsGsCsGsG ″ ″ ″ 25661 GoGoToAsGsGsAsGsAsCsGsGsCsGsAsTsGoCoGoG ″ ″ ″

[0244] TABLE 14 Dose Response of 20 Hour PMA-Induced neoHK Cells to TNF-α Antisense Oligonucleotides (ASOs) ASO Gene % protein % protein ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — —  100% — 14834 29 STOP  75 nM 91.2%  8.8% ″ ″ ″ 150 nM 42.0% 58.0% ″ ″ ″ 300 nM 16.9% 83.1% 21820 66 intron 1  75 nM 79.0% 21.0% ″ ″ ″ 150 nM 34.5% 65.5% ″ ″ ″ 300 nM 15.6% 84.4% 21823 69 intron 1  75 nM 79.5% 20.5% ″ ″ ″ 150 nM 31.8% 68.2% ″ ″ ″ 300 nM 16.2% 83.8% 21725 91 exon 4  75 nM 74.8% 25.2% ″ ″ ″ 150 nM 58.4% 41.6% ″ ″ ″ 300 nM 45.2% 54.8% 25655 91 exon 4  75 nM 112.0%  — ″ ″ ″ 150 nM 55.0% 45.0% ″ ″ ″ 300 nM 39.3% 60.7% 25656 91 exon 4  75 nM 108.3%  — ″ ″ ″ 150 nM 60.7% 39.3% ″ ″ ″ 300 nM 42.8% 57.2% 25660 91 exon 4  75 nM 93.2%  6.8% ″ ″ ″ 150 nM 72.8% 27.2% ″ ″ ″ 300 nM 50.3% 49.7%

[0245] TABLE 15 Dose Response of 20 Hour PMA-Induced neoHK Cells to TNF-α Antisense Oligonucleotides (ASOs) ASO Gene % protein % protein ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — —  100% — 14834 29 STOP  75 nM 44.9% 55.1% ″ ″ ″ 150 nM 16.3% 83.7% ″ ″ ″ 300 nM  2.2% 97.8% 21834 80 intron 3  75 nM 102.9%  — ″ ″ ″ 150 nM 24.5% 75.5% ″ ″ ″ 300 nM 19.1% 80.9% 21836 82 intron 3  75 nM 70.8% 29.2% ″ ″ ″ 150 nM 55.9% 44.1% ″ ″ ″ 300 nM 32.7% 67.3% 21732 98 exon 4  75 nM 42.4% 57.6% ″ ″ ″ 150 nM 34.9% 65.1% ″ ″ ″ 300 nM 15.4% 84.6% 25657 98 exon 4  75 nM 46.7% 53.3% ″ ″ ″ 150 nM 72.0% 28.0% ″ ″ ″ 300 nM 50.6% 49.4% 25658 98 exon 4  75 nM 83.7% 16.3% ″ ″ ″ 150 nM 56.6% 43.4% ″ ″ ″ 300 nM 36.9% 63.1% 25661 98 exon 4  75 nM 54.9% 45.1% ″ ″ ″ 150 nM 34.4% 65.6% ″ ″ ″ 300 nM  8.6% 91.4%

Example 7 Activity of Fully 2′-MOE Modified TNFα Antisense Oligonucleotides

[0246] A series of antisense oligonucleotides were synthesized targeting the terminal twenty nucleotides of each exon at every exon-intron junction of the TNFα gene. These oligonucleotides were synthesized as fully 2′-methoxyethoxy modified oligonucleotides. The oligonucleotide sequences are shown in Table 16. Oligonucleotide 12345 (SEQ ID NO. 106) is an antisense oligonucleotide targeted to the human intracellular adhesion molecule-1 (ICAM-1) and was used as an unrelated target control.

[0247] The oligonucleotides were screened at 50 nM and 200 nM for their ability to inhibit TNF-α mRNA levels, as described in Example 3. Results are shown in Table 17. Oligonucleotide 21794 (SEQ ID NO. 102) showed an effect at both doses, with greater than 75% inhibition at 200 nM. TABLE 16 Nucleotide Sequences of Human TNF-α Uniform 2′-MOE Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′ -> 3′) NO: ORDINATES² REGION³ 21792 AGGCACTCACCTCTTCCCTC 100 0972-0991 E1/I1 21793 CCCTGGGGAACTGTTGGGGA 101 1579-1598 I1/E2 21794 AGACACTTACTGACTGCCTG 102 1625-1644 E2/I2 21795 GAAGATGATCCTGAAGAGGA 103 1812-1831 I2/E3 21796 GAGCTCTTACCTACAACATG 104 1860-1879 E3/I3 21797 TGAGGGTTTGCTGGAGGGAG 105 2161-2180 I3/E4 12345 GATCGCGTCGGACTATGAAG 106 target control

[0248] TABLE 17 Dose Response of neoHK Cells to TNF-α Antisense 2′-MOE Oligonucleotides ASO Gene % mRNA % mRNA ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — — 100% — 12345 106 control  50 nM 121% — ″ ″ ″ 200 nM 134% — 13393  49 control  50 nM 110% — ″ ″ ″ 200 nM 112% — 14834  29 STOP  50 nM 92%  8% ″ ″ ″ 200 nM 17% 83% 21792 100 E1/I1  50 nM 105% — ″ ″ ″ 200 nM 148% — 21793 101 I1/E2  50 nM 106% — ″ ″ ″ 200 nM 172% — 21794 102 E2/I2  50 nM 75% 25% ″ ″ ″ 200 nM 23% 77% 21795 103 I2/E3  50 nM 79% 21% ″ ″ ″ 200 nM 125% — 21796 104 E3/I3  50 nM 56% 44% ″ ″ ″ 200 nM 150% — 21797 105 I3/E4  50 nM 90% 10% ″ ″ ″ 200 nM 128% —

Example 8 Mouse TNF-α Oligonucleotide Sequences

[0249] Antisense oligonucleotides were designed to target mouse TNF-α. Target sequence data are from the TNFα cDNA sequence published by Semon et al. (Nucleic Acids Res. 1987, 15, 9083-9084); Genbank accession number Y00467, provided herein as SEQ ID NO: 107. Oligonucleotides were synthesized primarily as phosphorothioate oligodeoxynucleotides. Oligonucleotide sequences es are shown in Table 18. Oligonucleotide 3082 (SEQ ID NO. 141) is an antisense oligodeoxynucleotide targeted to the human intracellular adhesion molecule-i (ICAM-1) and was used as an unrelated target control. Oligonucleotide 13108 (SEQ ID NO. 142) is an antisense oligodeoxynucleotide targeted to the herpes simplex virus type 1 and was used as an unrelated target control.

[0250] P388D1, mouse macrophage cells (obtained from American Type Culture Collection, Manassas, Va.) were cultured in RPMI 1640 medium with 15% fetal bovine serum (FBS) (Life Technologies, Rockville, Md.).

[0251] At assay time, cell were at approximately 90% confluency. The cells were incubated in the presence of OPTIMEM7 medium (Life Technologies, Rockville, Md.), and the oligonucleotide formulated in LIPOFECTIN7 (Life Technologies), a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water. For an initial screen, the oligonucleotide concentration was 100 nM in 3 μg/ml LIPOFECTIN7. Treatment was for four hours. After treatment, the medium was removed and the cells were further incubated in RPMI medium with 15% FBS and induced with 10 ng/ml LPS. mRNA was analyzed 2 hours post-induction with PMA.

[0252] Total mRNA was isolated using the TOTALLY RNA™ kit (Ambion, Austin, Tex.), separated on a 1% agarose gel, transferred to HYBOND™-N+ membrane (Amersham, Arlington Heights, Ill.), a positively charged nylon membrane, and probed. A TNF-α probe consisted of the 502 bp EcoRI-HindIII fragment from BBG 56 (R&D Systems, Minneapolis, Minn.), a plasmid containing mouse TNF-α cDNA. A glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe consisted of the 1.06 kb HindIII fragment from pHcGAP (American Type Culture Collection, Manassas, Va.), a plasmid containing human G3PDH cDNA. The fragments were purified from low-melting temperature agarose, as described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, 1989 and labeled with REDIVUE™ 32P-dCTP (Amersham Pharmacia Biotech, Piscataway, N.J.) and PRIME-A-GENE7 labeling kit (Promega, Madison, Wis.). mRNA was quantitated by a PhosphoImager (Molecular Dynamics, Sunnyvale, Calif.).

[0253] Secreted TNF-α protein levels were measured using a mouse TNF-α ELISA kit (R&D Systems, Minneapolis, Minn. or Genzyme, Cambridge, Mass.). TABLE 18 Nucleotide Sequences of Mouse TNF-α Phosphorothioate Oligodeoxynucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′ -> 3′) NO: ORDINATES² REGION 14846 GAGCTTCTGCTGGCTGGCTG 108 4351-4370 5′-UTR 14847 CCTTGCTGTCCTCGCTGAGG 109 4371-4390 5′-UTR 14848 TCATGGTGTCTTTTCTGGAG 110 4511-4530 AUG 14849 CTTTCTGTGCTCATGGTGTC 111 4521-4540 AUG 14850 GCGGATCATGCTTTCTGTGC 112 4531-4550 coding 14851 GGGAGGCCATTTGGGAACTT 113 5225-5244 junction 14852 CGAATTTTGAGAAGATGATC 114 5457-5476 junction 14853 CTCCTCCACTTGGTGGTTTG 115 5799-5818 junction 14854 CCTGAGATCTTATCCAGCCT 116 6540-6559 3′-UTR 14855 CAATTACAGTCACGGCTCCC 117 6927-6946 3′-UTR 15921 CCCTTCATTCTCAAGGCACA 118 5521-5540 junction 15922 CACCCCTCAACCCGCCCCCC 119 5551-5570 intron 15923 AGAGCTCTGTCTTTTCTCAG 120 5581-5600 intron 15924 CACTGCTCTGACTCTCACGT 121 5611-5630 intron 15925 ATGAGGTCCCGGGTGGCCCC 122 5651-5670 intron 15926 CACCCTCTGTCTTTCCACAT 123 5681-5700 intron 15927 CTCCACATCCTGAGCCTCAG 124 5731-5750 intron 15928 ATTGAGTCAGTGTCACCCTC 125 5761-5780 intron 15929 GCTGGCTCAGCCACTCCAGC 126 5821-5840 coding 15930 TCTTTGAGATCCATGCCGTT 127 5861-5880 coding 15931 AACCCATCGGCTGGCACCAC 128 5891-5910 coding 15932 GTTTGAGCTCAGCCCCCTCA 129 6061-6080 coding 15933 CTCCTCCCAGGTATATGGGC 130 6091-6110 coding 15934 TGAGTTGGTCCCCCTTCTCC 131 6121-6140 coding 15935 CAAAGTAGACCTGCCCGGAC 132 6181-6200 coding 15936 ACACCCATTCCCTTCACAGA 133 6211-6230 STOP 15937 CATAATCCCCTTTCTAAGTT 134 6321-6340 3′-UTR 15938 CACAGAGTTGGACTCTGAGC 135 6341-6360 3′-UTR 15939 CAGCATCTTGTGTTTCTGAG 136 6381-6400 3′-UTR 15940 CACAGTCCAGGTCACTGTCC 137 6401-6420 3′-UTR 15941 TGATGGTGGTGCATGAGAGG 138 6423-6442 3′-UTR 15942 GTGAATTCGGAAAGCCCATT 139 6451-6470 3′-UTR 15943 CCTGACCACTCTCCCTTTGC 140 6501-6520 3′-UTR  3082 TGCATCCCCCAGGCCACCAT 141 target control 13108 GCCGAGGTCCATGTCGTACGC 142 target control

[0254] Results are shown in Table 19. Oligonucleotides 14853 (SEQ ID NO. 115), 14854 (SEQ ID NO. 116), 14855 (SEQ ID NO. 117), 15921 (SEQ ID NO. 118), 15923 (SEQ ID NO. 120), 15924 (SEQ ID NO. 121), 15925 (SEQ ID NO. 122), 15926 (SEQ ID NO. 123), 15929 (SEQ ID NO. 126), 15930 (SEQ ID NO. 127), 15931 (SEQ ID NO. 128), 15932 (SEQ ID NO. 129), 15934 (SEQ ID NO. 131), 15935 (SEQ ID NO. 132), 15936 (SEQ ID NO. 133), 15937 (SEQ ID NO. 134), 15939 (SEQ ID NO. 136), 15940 (SEQ ID NO. 137), 15942 (SEQ ID NO. 139), and 15943 (SEQ ID NO. 140) gave better than 50% inhibition. Oligonucleotides 15931 (SEQ ID NO. 128), 15932 (SEQ ID NO. 129), 15934 (SEQ ID NO. 131), and 15943 (SEQ ID NO. 140) gave 75% inhibition or better. TABLE 19 Inhibition of Mouse TNF-α mRNA expression in P388D1 Cells by Phosphorothioate Oligodeoxynucleotides ISIS SEQ ID GENE TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION Induced — — 100%  0%  3082 141 control 129% — 13664  42 control 85% 15% 14846 108 5′-UTR 84% 16% 14847 109 5′-UTR 88% 12% 14848 110 AUG 60% 40% 14849 111 AUG 75% 25% 14850 112 coding 67% 33% 14851 113 junction 62% 38% 14852 114 junction 69% 31% 14853 115 junction 49% 51% 14854 116 3′-UTR 31% 69% 14855 117 3′-UTR 39% 61% 15921 118 junction 42% 58% 15922 119 intron 64% 36% 15923 120 intron 31% 69% 15924 121 intron 29% 71% 15925 122 intron 30% 70% 15926 123 intron 29% 71% 15928 125 intron 59% 41% 15929 126 coding 38% 62% 15930 127 coding 43% 57% 15931 128 coding 23% 77% 15932 129 coding 25% 75% 15933 130 coding 52% 48% 15934 131 coding 21% 79% 15935 132 coding 39% 61% 15936 133 STOP 35% 65% 15937 134 3′-UTR 45% 55% 15938 135 3′-UTR 76% 24% 15939 136 3′-UTR 33% 67% 15940 137 3′-UTR 38% 62% 15941 138 3′-UTR 54% 46% 15942 139 3′-UTR 42% 58% 15943 140 3′-UTR 25% 75%

Example 9 Dose Response of Antisense Phosphorothiaote Oligodeoxynucleotide Effects on Mouse TNFα mRNA Levels in P388D1 Cells

[0255] Four of the more active oligonucleotides from the initial screen were chosen for dose response assays. These include oligonucleotides 15924 (SEQ ID NO. 121), 15931 (SEQ ID NO. 128), 15934 (SEQ ID NO. 131) and 15943 (SEQ ID NO. 140). P388D1 cells were grown, treated and processed as described in Example 8. LIPOFECTIN7 was added at a ratio of 3 μg/ml per 100 nM of oligonucleotide. The control included LIPOFECTIN7 at a concentration of 6 μg/ml. Results are shown in Table 20. Each oligonucleotide tested showed a dose response effect with maximal inhibition about 70% or greater and IC₅₀ values less than 50 nM. TABLE 20 Dose Response of LPS-Induced P388D1 Cells to TNF-α Antisense Phosphorothioate Oligodeoxynucleotides (ASOs) ASO Gene % mRNA % mRNA ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — — 100% — 13108 142 control  25 nM 68% 32% ″ ″ ″  50 nM 71% 29% ″ ″ ″ 100 nM 64% 36% ″ ″ ″ 200 nM 75% 25% 15924 121 intron  25 nM 63% 37% ″ ″ ″  50 nM 49% 51% ″ ″ ″ 100 nM 36% 64% ″ ″ ″ 200 nM 31% 69% 15931 128 coding  25 nM 42% 58% ″ ″ ″  50 nM 30% 70% ″ ″ ″ 100 nM 17% 83% ″ ″ ″ 200 nM 16% 84% 15934 131 coding  25 nM 37% 63% ″ ″ ″  50 nM 26% 74% ″ ″ ″ 100 nM 13% 87% ″ ″ ″ 200 nM 13% 87% 15943 140 3′-UTR  25 nM 38% 62% ″ ″ ″  50 nM 38% 62% ″ ″ ″ 100 nM 16% 84% ″ ″ ″ 200 nM 16% 84%

Example 10 Design and Testing of 2′-O-methoxyethyl (Deoxy Gapped) TNFα Antisense Oligonucleotides on TNFα Levels in P388D1 Cells

[0256] Oligonucleotides having SEQ ID NO: 128, SEQ ID NO: 131, and SEQ ID NO: 140 were synthesized as uniformly phosphorothioate oligodeoxynucleotides or mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. The sequences and the oligonucleotide chemistries are shown in Table 21. All 2′-MOE cytosines were 5-methyl-cytosines. Oligonucleotides were screened as described in Example 8. Results are shown in Table 22. All the oligonucleotides tested, except oligonucleotide 16817 (SEQ ID NO. 140) showed 44% or greater inhibition of TNF-α mRNA expression. Oligonucleotides 16805 (SEQ ID NO: 131), 16813 (SEQ ID NO: 140), and 16814 (SEQ ID NO: 140) showed greater than 70% inhibition. TABLE 21 Nucleotide Sequences of Mouse 2′-O-methoxyethyl (deoxy gapped) TNF-α Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES² REGION 15931 AsAsCsCsCsAsTsCsGsGsCsTsGsGsCsAsCsCsAsC 128 5891-5910 coding 16797 AoAoCoCsCsAsTsCsGsGsCsTsGsGsCsAsCoCoAoC ″ 5891-5910 coding 16798 AsAsCsCsCsAsTsCsGsGsCsTsGsGsCsAsCsCsAsC ″ 5891-5910 coding 16799 AoAoCoCoCsAsTsCsGsGsCsTsGsGsCsAoCoCoAoC ″ 5891-5910 coding 16800 AsAsCsCsCsAsTsCsGsGsCsTsGsGsCsAsCsCsAsC ″ 5891-5910 coding 16801 AoAoCoCoCoAoToCoGsGsCsTsGsGsCsAsCsCsAsC ″ 5891-5910 coding 16802 AsAsCsCsCsAsTsCsGsGsCsTsGsGsCsAsCsCsAsC ″ 5891-5910 coding 16803 AsAsCsCsCsAsTsCsGsGsCsToGoGoCoAoCoCoAoC ″ 5891-5910 coding 16804 AsAsCsCsCsAsTsCsGsGsCsTsGsGsCsAsCsCsAsC ″ 5891-5910 coding 15934 TsGsAsGsTsTsGsGsTsCsCsCsCsCsTsTsCsTsCsC 131 6121-6140 coding 16805 ToGoAoGsTsTsGsGsTsCsCsCsCsCsTsTsCoToCoC ″ 6121-6140 coding 16806 TsGsAsGsTsTsGsGsTsCsCsCsCsCsTsTsCsTsCsC ″ 6121-6140 coding 16807 ToGoAoGoTsTsGsGsTsCsCsCsCsCsTsToCoToCoC ″ 6121-6140 coding 16808 TsGsAsGsTsTsGsGsTsCsCsCsCsCsTsTsCsTsCsC ″ 6121-6140 coding 16809 ToGoAoGoToToGoGoTsCsCsCsCsCsTsTsCsTsCsG ″ 6121-6140 coding 16810 TsGsAsGsTsTsGsGsTsCsCsCsCsCsTsTsCsTsCsC ″ 6121-6140 coding 16811 TsGsAsGsTsTsGsGsTsCsCsCoCoCoToToCoToCoC ″ 6121-6140 coding 16812 TsGsAsGsTsTsGsGsTsCsCsCsCsCsTsTsCsTsCsC ″ 6121-6140 coding 15943 CsCsTsGsAsCsCsAsCsTsCsTsCsCsGsTsTsTsGsC 140 6501-6520 3′-UTR 16813 CoCoToGsAsCsGsAsCsTsCsTsCsCsCsTsToToGoC ″ 6501-6520 3′-UTR 16814 CsCsTsGsAsCsCsAsCsTsCsTsCsCsCsTsTsTsGsC ″ 6501-6520 3′-UTR 16815 CoCoToGoAsCsCsAsCsTsCsTsCsCsCsToToToGoC ″ 6501-6520 3′-UTR 16816 CsCsTsGsAsCsCsAsCsTsCsTsCsCsCsTsTsTsGsC ″ 6501-6520 3′-UTR 16817 CoCoToGoAoCoCoAoCsTsCsTsCsCsCsTsTsTsGsC ″ 6501-6520 3′-UTR 16818 CsCsTsGsAsCsCsAsCsTsCsTsCsCsCsTsTsTsGsC ″ 6501-6520 3′-UTR 16819 CsCsTsGsAsCsCsAsCsTsCsToCoCoCoToToToGoC ″ 6501-6520 3′-UTR 16820 CsCsTsGsAsCsCsAsCsTsCsTsCsCsCsTsTsTsGsC ″ 6501-6520 3′-UTR

[0257] TABLE 22 Inhibition of mouse TNF-α mRNA expression in P388D1 Cells by 2′-O-methoxyethyl (deoxy gapped) Oligonucleotides ISIS SEQ ID GENE TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION induced — — 100%  0% 13108 142 control 87% 13% 15934 131 coding 28% 72% 16797 128 coding 33% 67% 16798 ″ coding 34% 66% 16799 ″ coding 56% 44% 16800 ″ coding 35% 65% 16801 ″ coding 34% 66% 16802 ″ coding 38% 62% 16803 ″ coding 35% 65% 16804 ″ coding 39% 61% 16805 131 coding 29% 71% 16806 ″ coding 31% 69% 16807 ″ coding 46% 54% 16808 ″ coding 43% 57% 16809 ″ coding 33% 67% 16810 ″ coding 37% 63% 16811 ″ coding 40% 60% 16812 ″ coding 31% 69% 16813 140 3′-UTR 28% 72% 16814 ″ 3′-UTR 28% 72% 16815 ″ 3′-UTR 46% 54% 16816 ″ 3′-UTR 49% 51% 16817 ″ 3′-UTR 172% — 16818 ″ 3′-UTR 34% 66% 16819 ″ 3′-UTR 51% 49% 16820 ″ 3′-UTR 44% 56%

Example 11 Effect of TNFα Antisense Oligonucleotides in a Murine Model for Non-Insulin-dependent Diabetes Mellitus

[0258] The db/db mouse model, a standard model for non-insulin-dependent diabetes mellitus (NIDDM; Hotamisligil, G. S., et al., Science, 1993, 259, 87-90), was used to assess the activity of TNF-α antisense oligonucleotides on blood glucose levels and TNFα mRNA levels in whole mice. These mice have elevated blood glucose levels and TNF-α mRNA levels compared to wild type mice. Female db/db mice and wild-type littermates were purchased from Jackson Laboratories (Bar Harbor, Me.). The effect on oligonucleotide 15931 (SEQ ID NO. 128) on blood glucose levels was determined. For determination of TNF-α mRNA levels, oligonucleotide 15931 (SEQ ID NO. 128), a uniformly modified phosphorothioate oligodeoxynucleotide, was compared to oligonucleotide 25302 (SEQ ID NO. 128), a mixed phosphorothioate/phosphodiester chimeric oligonucleotide having regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. The sequences and chemistries are shown in Table 23. Oligonucleotide 18154 (SEQ ID NO. 143) is an antisense mixed phosphorothioate/phosphodiester chimeric oligonucleotide, having regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides, targeted to the human vascular cell adhesion molecule-1 (VCAM-1) and was used as an unrelated target control. TABLE 23 Nucleotide Sequence of TNF-α Antisense Oligonucleotide TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′ -> 3′) NO: ORDINATES² REGION 15931 AACCCATCGGCTGGCACCAC 128 5891-5910 coding 25302 AACCCATCGGCTGGCACCAC 128 5891-5910 coding 18154 TCAAGCAGTGCCACCGATCC 143 target control

[0259] db/db mice, six to ten weeks old, were dosed intraperitoneally with oligonucleotide every other day for 2 weeks at 10 mg/kg. The mice were fasted for seven hours prior to administration of the oligonucleotide. The mice were bled via retro orbital sinus every other day, and glucose measurements were performed on the blood. Results are shown in Table 24. Oligonucleotide 15931 (SEQ ID NO. 128) was able to reduce blood glucose levels in db/db mice to levels comparable with wild type mice. Food intake between wild type mice, treated and untreated, did not differ. Food intake between db/db mice, treated and untreated, although higher than wild type mice, did not differ significantly.

[0260] Samples of the fat (adipose) tissue from the inguinal fat pads were taken for RNA extraction. RNA was extracted according to Current Protocols in Molecular Biology, 1997, Ausubel, F., et al. ed., John Wiley & Sons. RNA was purified using the RNA clean up procedure of the RNEASY7 Mini kit (Qiagen, Valencia, Calif.). TNF-α mRNA levels were measured using the RIBOQUANT7 kit (PharMingen, San Diego, Calif.) with 15 μg of RNA per lane. The probe used was from the mCK-3b Multi-Probe Template set (PharMingen, San Diego, Calif.) labeled with [α³²P]UTP (Amersham Pharmacia Biotech, Piscataway, N.J.). Results are shown in Table 25. Both oligonucleotide 15931 (SEQ ID NO. 128) and 25302 (SEQ ID NO. 128) were able to reduce TNFα levels in fat, with 25302 (SEQ ID NO. 128) reducing TNF-α to nearly wild-type levels. TABLE 24 Level of Blood Glucose in Normal and db/db Mice After Treatment with TNF-α Antisense Oligonucleotides Mouse ASO Gene Time blood glucose Strain ISIS # SEQ ID NO: Target (days) (mg/dL) wild type — — — 1 140 ″ 15931 128 coding ″ 138 db/db — — — 1 260 ″ 15931 128 coding ″ 254 wild type — — — 9 175 ″ 15931 128 coding ″ 163 db/db — — — 9 252 ″ 15931 128 coding ″ 128

[0261] TABLE 25 Level of TNF-α mRNA in Fat of db/db Mice After Treatment with TNF-α Antisense Oligonucleotides GENE ISIS SEQ ID TARGET % mRNA No: NO: REGION EXPRESSION wt saline — — 100% db/db saline — — 362% 18154 142 control 130% 15931 128 coding 210% 25302 128 coding 417%

Example 12 Effect of TNF-α Antisense Oligonucleotides in a Murine Model for Rheumatoid Arthritis

[0262] Collagen-induced arthritis (CIA) was used as a murine model for arthritis (Mussener, A., et al., Clin. Exp. Immunol., 1997, 107, 485-493). Female DBA/1LacJ mice (Jackson Laboratories, Bar Harbor, Me.) between the ages of 6 and 9 weeks were used to assess the activity of TNF-α antisense oligonucleotides. In all studies, 10 mice were used per treatment group.

[0263] On day 0, the mice were immunized at the base of the tail with 100 μg of bovine type II collagen which was emulsified in Complete Freund's Adjuvant (CFA). On day 7, a second booster dose of collagen was administered by the same route. On day 14, the mice were injected subcutaneously with 100 μg of LPS. Oligonucleotide was administered intraperitoneally (bolus) three times per week, starting on day 0, for the duration of the 7 week study at the indicated doses. The anti-TNF-α mAb (MM350D, Endogen, Woburn, Mass.) was administered intraperitoneally at 2 mg/kg once per week, starting on day 0. This antibody was formulated free of preservatives and carrier, and had an endotoxin level of 9.06 EU/mg.

[0264] Weights were recorded weekly. Mice were inspected daily for the onset of CIA, characterized by erythema and edema. Upon the onset of the disease, an assessment chart for each animal was started. Paw widths are rear ankle widths of affected and unaffected joints were measured three times a week using a constant tension caliper. Limbs were clinically evaluated and graded on a scale from 0-4, where o=normal, 1=one digit swollen, 2=inflammation present in more than one digit, 3=joint distortion with or without inflammation, and 4=ankylosis as detected by joint manipulation. The progression of all measurements recorded to day 50. On day 50, animals were euthanized by cervical dislocation. All paws were removed and fixed in 10% neutral buffered formalin, from which histopathology slides were prepared.

[0265] Arthritis was classified into four stages based on histological evaluation of the degres of inflammation, cartilage damage, pannus formation, bone erosion, osteolysis, fibrosis and ankylosis. Stage I is described by inflammatory cell infiltration in the tissues surrounding the joint and/or superficial layers of the synovium. Stage II is described by pannus formation with damage to the superficial layers of the cartilage. Stage III is described by subchondral bone erosion with some degree of osteoloysis. Stage IV is described by severe destruction of cartilage and bone with areas of fibrosis and/or bony ankylosis. The clinical data was analyzed for differences in the incidence of disease, the onset of disease and the severity of the disease. Descriptive statistics and an analysis of variance (ANOVA) were performed. If a statistically significant difference was detected, a Dunnett's test was performed.

[0266] Two independent studies, which differed in dose range, showed that mice treated with ISIS 25302 had a reduced incidence of arthritis (FIGS. 1A-1B). The two dose ranges were 0.03 to 3.0 mg/kg (low range, FIG. 1A), and 2.5 to 20 mg/kg (high range, FIG. 1B). The lowest incidence of disease was observed in mice treated at doses of 3.0 (22%) and 2.5 mg/kg (38%) of ISIS 25302 respectively, as compared to the vehicle control incidence of 88% in both studies. No further reduction in the incidence of disease occurred in mice treated at higher doses. The onset of disease was delayed in groups treated with ISIS 25302, but varied between experiments (Table 1). The severity of the disease and the percent affected paws were also reduced by treatment with ISIS 25302. Best effects on these clinical outcomes were observed at 3.0 mg/kg in the low dose range study, and 2.5 and 20 mg/kg in the high dose range study.

[0267] Treatment of mice with the eight mismatch control, ISIS 30782 (5′CACCAAGCTGCGGTCCCCAA 3′; SEQ ID NO: 502), yielded variable results between the low dose (Table 26A) and high dose (Table 26B) range studies. In the low dose range study, the one group treated with the control oligonucleotide, at a dose of 3.0 mg/kg, showed comparable improvements in the clinical outcome in comparison to the group treated with the anti-TNF-α oligonucleotide of equivalent dose. In contrast, the eight mismatch control oligonucleotide had minimal effects on the clinical outcome in the high dose range study, at doses of 2.5, 5.0, and 10 mg/kg; but did show effects in the clinic at the highest dose of 20 mg/kg. TABLE 26A % % Dose inci- Day of Severity affected Treatment Schedule (mg/kg) dence onset (″SEM) paws Vehicle 3x/wk — 88 18.1″0.7 7.1″2.1 59 ISIS 25302 3x/wk 0.03 70 18.6″1.1 3.1″1.2 28 ISIS 25302 3x/wk 0.1 70 17.6″0.2 3.5″1.5 30 ISIS 25302 3x/wk 0.3 44 21.5″4.5 2.9″1.4 25 ISIS 25302 3x/wk 1.0 67 21.0″3.6 3.4″1.0 36 ISIS 25302 3x/wk 3.0 22 21.5″3.5 1.2″0.8 14 TNF mAb 1x/wk 2.0 30 28.0″1.5 1.3″0.7 8.3 8MM ctrl 3x/wk 3.0 22 17.5″0.5 1.0″0.7 8.3

[0268] TABLE 26B % % Dose inci- Day of Severity affected Treatment Schedule (mg/kg) dence Onset (″SEM) paws Vehicle 3x/wk — 88 17.6″0.4 6.0″1.6 53 ISIS 25302 3x/wk 2.5 38 28.3″10.8 2.1″1.5 19 ISIS 25302 3x/wk 5.0 50 23.2″5.7 4.5″1.7 40 ISIS 25302 3x/wk 10 44 17.0″0.4 4.0″1.7 33 ISIS 25302 3x/wk 20 56 23.8″5.1 2.2″1.4 19 8MM ctrl 3x/wk 2.5 71 17.4″0.7 6.3″2.2 57 8MM ctrl 3x/wk 5.0 86 20.7″3.1 6.6″2.1 57 8MM ctrl 3x/wk 10 80 18.0″0.6 6.4″1.5 55 8MM ctrl 3x/wk 20 44 19.5″1.6 1.7″1.3 17

[0269] In both tables, the incidence is the number of mice with at least one affected paw/total number of mice per group. Severity is the total clinical score/total number of mice in the group. Percent affected paws=(number of affected paws at termination/total number of paws in group)×100. 8MM ctrl=eight mismatch control (ISIS 30782).

[0270] Efficacy of ISIS 25302 (3 mg/kg, three times per week) was found to be comparable to that of an anti-TNF-α mAb (2 mg/kg, once per week) as described in Table 26A. The disease incidence in mice treated with ISIS 25302 was 22% versus 30% for the group treated with the anti-TNF-α mAb. Disease severity and percent affected paws were also reduced to a similar degree in the 3 mg/kg ISIS 25302 and anti-TNF-α mAb treated groups.

[0271] Mice treated with the anti-mTNF-α oligonucleotide, ISIS 25302, showed an improvement in the disease outcome when treated three times per week starting on the initial day of collagen-induction. Reduction of symptoms by the ISIS 25302 was dose dependent, and showed equivalent effects when compared to mice treated with an anti-TNF-α monoclonal antibody once per week from the time of collagen-induction. Histological evaluation of the joints showed a reduction in the incidence and severity of arthritic lesions in mice treated with ISIS 25302, but to a lesser extent than those mice treated with the anti-TNF-α mAb.

[0272] The efficacy of ISIS 25302 compares favorably to other anti-TNF biological agents which have been evaluated in the “classical” CIA model. For instance, treatment of mice with the recombinant human TNF receptor FC fusion protein prior to onset of disease resulted in a 28% incidence of disease as compared to 86% incidence in the saline control treated animals (Wooley, J. Immunol. 151:6602-6607, 1993). In addition, preventative treatment by an anti-TNF-α antibody in the “classical” model showed 40% reduction in paw swelling in the clinic, as well as reduction in histopathological severity (Williams, Proc. Natl. Acad Sci. U.S.A. 89:9784-9788, 1992).

[0273] A marked difference was observed between the two independent studies of ISIS 25302 in this model of CIA, with respect to responsiveness of the animals to oligonucleotide treatment. Mice were more responsive to oligonucleotide treatment in the low dose range study. This responsiveness was reflected in the histological results, where all oligonucleotide treated groups showed a notable reduction in paw incidence in comparison to the vehicle group. In comparison to the high dose study, mice in the low dose study overall displayed a lower percentage of paws with arthritic changes at the histological level.

[0274] In conclusion, evaluation of ISIS 25302 in the accelerated CIA model has shown that an anti-TNF-α oligonucleotide provides an alternative approach to treatment of related human disease indications. Potential advantages of the antisense oligonucleotide therapeutic approach, over the current anti-arthritic (biological) agents, include ease of administration and a lower potential for adverse effects from long term usage.

Example 13 Effect of TNF-α Antisense Oligonucleotides in a Murine Model for Contact Sensitivity

[0275] Contact sensitivity is a type of immune response resulting from contact of the surface of the skin with a sensitizing chemical. A murine model for contact sensitivity is widely used to develop therapies for chronic inflammation, autoimmune disorder, and organ transplant rejection (Goebeler, M., et al., Int Arch. Allergy Appl. Immunol., 1990, 93, 294-299). One example of such a disease is atopic dermatitis. Female Balb/c mice between the ages of 8 and 12 weeks are used to assess the activity of TNFα antisense oligonucleotides in a contact sensitivity model.

[0276] Balb/c mice receive injections of oligonucleotide drug in saline via i.v. injection into the tail vein. The abdomen of the mice is shaved using an Oster hair clipper. The animals are anesthetized using isoflurane, and 25 μl of 0.2% 2,4-dinitrofluorobenzene (DNFB) in 4:1 acetone:olive oil is applied to the shaved abdomen two days in a row. After five days, 10 ml of 0.2% DNFB in the same vehicle is applied to the right ear. After each exposure, the mouse is suspended in air for two minutes to allow the DNFB to absorb into the skin. 24 and 48 hours after application of DNFB to the ear, the ear thickness is measured using a micrometer. Inflammation (dermatitis) is indicated by a ranked thickening of the ear. Thickness of the treated ear is compared to untreated (contralateral) ear thickness.

Example 14 Effect of TNFα Antisense Oligonucleotides in an IL10(−/−) Murine Model for Colitis

[0277] The effects of antisense oligonucleotide-inhibition of TNFα was studied in the IL-10^(−/−) mouse model of colitis. IL10 deficient mice IL-10^(−/−) display some of the features that are observed in Crohn's disease such as discontinuous lesions throughout the gastrointestinal tract and have a cytokine profile that is characteristic of a Th1 immune response. Unlike Crohn's disease, however, IL-10^(−/−) mice show a marked crypt hyperplasia and absence of fissures and fistulas. In addition, IL-10^(−/−) mice have elevated levels of TNF-α expression.

[0278] Animals were treated in a prophylactic manner with one of four doses of ISIS 25302 or ISIS. Dosing extended from two weeks of age, before the development of colitis, to eight weeks of age, a time at which IL-10^(−/−) mice typically exhibit advanced stages of colitis. Colitis was assessed by histological evaluation at the conclusion of the study, and the basal and induced secretion of IFN-γ and TNF-α from colon organ culture supernatants was also measured at that time.

[0279] Homozygous Interleukin-10 gene-deficient mice, generated on a 129 Sv/Ev background, and 129 Sv/Ev controls were housed under specific pathogen-free conditions. Mice were housed in micro-isolator cages with tight-fitting lids containing spun-polyester fiber filters. Mice were injected every other day with either ISIS 25302 or ISIS 30782 (the 8 mismatch control) at 0.01, 0.1, 1.0, and 10 mg/kg from 2-8 weeks of age via subcutaneous injection.

[0280] Animals were sacrificed using sodium pentobarbitol (160 mg/kg). Whole colons were harvested, cut lengthwise, and fixed in 10% phosphate-buffered formalin, paraffin-embedded, sectioned at 4 μm, and stained with haematoxylin and eosin for light microscopic examination. The slides were reviewed independently by a pathologist in a blinded fashion and assigned a histological score for intestinal inflammation (Table 27). The total histological score represents the numerical sum of the four scoring criteria: mucosal ulceration, epithelial hyperplasia, lamina propria mononuclear cell infiltration, and lamina propria neutrophilic infiltration. TABLE 27 Mucosal Epithelial LP mononuclear LP neutrophil Score ulceration hyperplasia infiltration infiltrate 0 Normal Normal Normal Normal 1 Surface Mild Slight increase Slight inflammation increase 2 Erosions Moderate Marked increase Marked increase 3 Ulcerations Pseudopolyps

[0281] Colonic organ cultures were prepared from IL-10 gene-deficient mice treated for six weeks. Due to the patchy nature of colitis in IL-10 gene-deficient mice, whole colons were removed, cut lengthwise, flushed with PBS, and resuspended in tissue culture plates (Falcon 3046; Becton Dickinson Labware, Lincoln Park, N.J.) in RPMI-1640 medium supplemented with 10% fetal calf serum, 50 mM 2-mercaptoethanol, penicillin (100 U/mL), and streptomycin (100 U/mL). Cultures were incubated at 37° C. in 5% CO₂. After 24 hours in the absence (basal) or presence of 10 μg/ mL LPS (E. coli, 0111:B4, Sigma), supernatants were harvested and stored at −70° C. for analysis of cytokine levels. TNF-α and IFN-γ levels in cell supernatants were measured using ELISA kits purchased from Biosource Cytoscreen (Montreal, Quebec).

[0282] Differences between treatment groups were evaluated by analysis of variance (ANOVA). Single arm analysis was performed by the Dunnett's test (SAS Institute Inc., Cary N.C.)

[0283] Over the 6-week treatment period, all treatment groups of IL-10 deficient mice gained weight at a similar rate (data not shown). At 8 weeks of age, IL-10^(−/−) mice displayed a patchy distribution of transmural acute and chronic inflammation, extensive mucosal ulceration, and epithelial hyperplasia. Table 28 shows the histological scores for colon tissue from IL-10^(−/−) mice treated with saline (vehicle), ISIS 25302 or ISIS 30782 (8MM ctrl) from 2 to 8 weeks of age at the indicated doses (n=6). The “total” histological score is the summation of the scores determined for each of the four histological parameters: mucosal ulceration, epithelial hyperplasia, lamina propia (LP) mononuclear cell infiltration, and lamina propria neutrophilic infiltration. Mice receiving the 0.1 mg/kg dose of the anti-TNF-α oligonucleotide, ISIS 25302, demonstrated a marked improvement in their mucosal architecture, which was statistically significant (p<0.05) in comparison to the Vehicle (saline) group (FIG. 2). No other group showed a significant histological difference in comparison to Vehicle. TABLE 28 Mononuclear Neutrophil Treatment Score Mucosal ulceration Mucosal hyperplasia infiltrate infiltrate Total Saline Mean 1.00 1.83 2.00 1.83 6.67 Std. 0.89 0.41 0.00 0.41 1.21 Dev. 0.01 mg/kg Mean 0.50 1.50 1.50 1.50 5.00 ISIS 25302 Std. 0.55 0.55 0.55 0.55 0.63 Dev.  0.1 mg/kg Mean 0.50 0.83 1.33 1.00 3.67 ISIS 25302 Std. 0.55 0.41 0.52 0.63 0.52 Dev.   1 mg/kg Mean 0.67 2.00 1.67 1.67 6.00 ISIS 25302 Std. 1.21 0.89 0.52 0.52 2.61 Dev.   10 mg/kg Mean 1.17 1.83 1.83 1.17 6.00 ISIS 25302 Std. 1.47 0.98 0.41 0.75 2.83 Dev. 0.01 mg/kg Mean 0.83 1.83 1.33 1.67 5.67 8MM ctrl Std. 1.17 0.75 0.52 0.52 2.58 Dev.  0.1 mg/kg Mean 1.00 1.67 1.33 1.17 5.17 8MM ctrl Std. 0.63 0.52 0.52 0.52 0.63 Dev.   1 mg/kg Mean 0.67 1.67 1.33 1.33 5.00 8MM ctrl Std. 0.52 0.52 0.52 0.52 0.63 Dev.   10 mg/kg Mean 0.83 2.00 1.33 1.50 5.67 8MM ctrl Std. 1.17 0.63 0.52 0.55 2.25 Dev.

[0284] Reduction of secreted TNFα protein levels was observed in colon tissue isolated from mice treated every other day with 0.1 mg/kg of ISIS 25302 under both basal (FIG. 3A) and LPS-induced (FIG. 3B) conditions. IFN-γ protein secretion from the isolated colon tissue was also reduced in the 0.1 mg/kg ISIS 25302 treated group relative to the saline treated group under both culture conditions (basal, FIG. 4A; LPS-induced, FIG. 4B). These effects were sequence specific, as the eight base mismatch oligonucleotide at the same dose of 0.1 mg/kg had no effect on basal or LPS-induced TNF-α protein secretion, or LPS-induced IFN-γ secretion.

[0285] Although treatment of IL-10^(−/−) mice with an antisense oligonucleotide targeted to TNF-α had no effect on the rate at which these animals gained weight, anti-TNF-α oligonucleotide treatment did have effects on several key disease parameters. Most importantly, antisense treatment at a relatively low dose (0.1 mg/kg) significantly reduced histological signs of colitis in the mice. This included reductions in mucosal ulceration, mucosal hyperplasia, and infiltrations of mononuclear cells and neutrophils into the lamina propria of the colon. These effects were not seen with the eight-base mismatch control oligonucleotide, ISIS 30782, which indicated that the effect was sequence specific.

[0286] The histological improvement is most likely due to specific reduction in TNFα protein levels with antisense treatment. Both the basal and LPS-induced secretion of TNFα from colons of mice treated with 0.1 mg/kg of ISIS 25302 were reduced, while the control oligonucleotide had no effect. A decrease in basal and induced IFN-γ levels also occurred in the mice treated with 0.1 mg/kg ISIS 25302. Interruption of the proinflammatory cytokine cascade by inhibition of TNF-α expression may have abrogated the recruitment and activation of CD4+ T cells that produce IFN-γ. TNF-α is known to activate expression of key inflammatory intermediates which promote this process, including expression of cell adhesion molecules, chemokines, and other proinflammatory cytokines (Zhang et al. “Tumor necrosis factor” in The Cytokine Handbook, 3rd ed., Academic Press Ltd., pp. 517-547; van Deventer, Gut 40:443-448, 1997).

[0287] A biphasic response to the anti-TNF-α oligonucleotide was observed in this genetically engineered mouse model of colitis, where optimal efficacy of the anti-TNF-α oligonucleotide occurred at the mid range dose of 0.1 mg/kg. Treatment at the higher doses of 1.0 and 10 mg/kg resulted in complete loss of efficacy, as observed histologically and by cytokine expression levels. The basis of this response may lie in the undefined roles of the pro- and anti-inflammatory cytokines in the absence of IL-10; and/or the pharmacokinetics and mechanism of action of the oligonucleotide.

[0288] In conclusion, ISIS 25302 reduced TNF-α expression levels in a dose and sequence-dependent manner in the IL-10 deficient mice. Specific reduction of this proinflammatory molecule diminished the pathological features associated with the intestinal injury and inflammation which occurs in the absence of IL-10 in these mice. The results from this mouse model of colitis indicate that antisense oligonucleotides to TNF-α represent a new treatment of Crohn's disease in man.

Example 15 Effect of TNFα Antisense Oligonucleotides in a DSS-induced Murine Model for Colitis

[0289] The pathological features of DSS-induced colitis in mice are similar in many respects to human ulcerative colitis (UC) (Table 29). This model is characterized by ulceration, epithelial damage, mucosal or transmural inflammatory infiltrate, and lymphoid hyperplasia of the colon. These effects are attributed to inappropriate macrophage function, alterations of the lumina bacteria, and the direct toxic effects of DSS on the colonic epithelium (Okayasu, Gastroenterol. 98:694-702, 1990). Both acute and chronic colitis may be studied in this model, by alteration of the DSS administration schedule (Okayasu, 1990, supra.; Cooper et al., Lab. Invest. 69:238-249, 1993). The efficacy of an anti-TNF-α mAb has been shown in both the acute and chronic model of DSS-induced colitis (Murthy et al., Aliment. Pharmacol. Ther. 13:251-260, 1999; Kojougaroff et al., Clin. Exp. Immunol. 107:353-358, 1997), as well as efficacy of an antisense oligonucleotide to ICAM-1 in the acute model of DSS-induced colitis (Bennett et al., J. Pharmacol. Exp. Ther. 280:988-1000, 1997). TABLE 29 Ulcerative DSS-induced Feature Crohn's colitis colitis Location GI tract Colon Colon Depth Transmural Mucosal Mucosal Extent Discontinuous Continuous Continuous Symptoms Non-bloody Bloody BD, no diarrhea, diarrhea, no fistula fistula fistula Granuloma Yes No No Genetic Yes Yes Yes Microbial Yes Yes Yes Immunological Yes Yes Yes Inflammation Transmural Epithelium Epithelium TNF-α Elevated Elevated Elevated

[0290] ISIS 25302 was evaluated for efficacy in both the acute and chronic models of DSS-induced colitis. ISIS 25302 is similar in design to the human anti-TNF-α oligonucleotide, ISIS 104838, with respect to the phosphorothioate modified backbone, methylated cytosine residues, and modification of each of the five 5′ and 3′ sugar residues with 2′-O-(2-methoxyethyl).

[0291] Female Swiss-Webster mice, 7 to 8 weeks of age weighing 25 to 30 grams, were obtained from Taconic or Jackson Laboratory. The animals were housed at 22° C. and 12 hours of dark and light cycles. Mouse chow and water were made available ab libitum.

[0292] Female Swiss-webster mice (n=2) were intravenously injected with 20 mg/kg of ISIS 13920 in saline or with saline alone on day 1, 3, and 5 of the acute DSS-induced colitis protocol as described below. ISIS 13920 is a fully modified phosphorothioate oligodeoxynucleotide, 5′ TCCGTCATCGCTCCTCAGGG 3′ (SEQ ID NO: 503), with 2′-O-(2-methoxyethyl) modified indicated by underline. This oligonucleotide is directed to the human ras-Ha gene. Two additional groups (n=2) of normal mice (no DSS) were subjected to the same oligonucleotide administration protocol. Mice were sacrificed on day 7. Colons were removed, trimmed longitudinally, fixed in 10% neutral buffered formaldehyde, and processed through paraffin. Four micron sections were cut from paraffin-embedded tissues, and deparaffinized by standard histological procedures. Endogenous tissue peroxidase activity was quenched with Peroxidase Blocking Reagent (DAKO; Carpenteria, Calif.) for 10 min at room temperature (r.t.). Tissue was treated with proteinase K (DAKO) for 10 min at r.t. to make it permeable for staining. After blocking with normal donkey serum (Jackson Laboratory; Bar Harbor, Me.), the sections were incubated for 45 min at r.t. with the 2E1-B5 anti-oligonucleotide mAb (Butler et al., Lab. Invest., 77:379-388, 1997). Sections were rinsed with PBS and then incubated with peroxidase conjugated rabbit anti-mouse IgG1 (Zymed Laboratories; San Francisco, Calif.) diluted 1:200 for 30 min at r.t. Slides were washed thoroughly with PBS and then stained for peroxidase activity by addition of 3,3′-diamino-benzidine (DAKO) for 5 min at r.t.

[0293] Mice received 4% dextran sodium sulfate (MW 40,000, ICN Biomedicals, Inc., Aurora Ohio) in double distilled water ad libitum from day 0 until day 5 to induce colitis. On day 5, the 4% DSS was replaced with plain drinking water.

[0294] Mice were first weighed and randomized into groups of seven or eight animals. Mice were administered oligonucleotide every other day (q2d) by i.v. or s.c. injection at the indicated doses from day −2 to day 6. The vehicle group was administered 1 mL/kg 0.9% saline (Baxter Healthcare Corporation, Deerfield, Ill.) under a similar treatment protocol.

[0295] Disease activity index was calculated on day 7 based on the summation of the weight, hemoccult, and stool consistency scores (Table 30). Mice were weighed initially on day 0, and then every day beginning on day 3 until time of sacrifice. The stool consistency from each mouse was evaluated daily by visible appearance, beginning on day 3. On the day of sacrifice, day 7, stool from each mouse was evaluated for occult blood using the Hemoccult test (SmithKline Diagnostics, Inc., San Jose Calif.). After sacrifice, the colon was removed from the ileocecal junction to the anal verge. The entire colon was then measured and observed for gross changes in the appearance of the mucosa, the total length of the colon was noted, and sections of the colon were dissected for histopathological evaluation. TABLE 30 Score Weight loss Stool consistency Hemoccult 0 None Normal Negative 1  1-5%  2  6-10% Loose stool Positive 3 11-15% 4 >15% Diarrhea Gross bleeding

[0296] Mice were first weighed and randomized into groups of eight to ten animals. Chronic colitis was induced by giving the mice 4% DSS in their drinking water for two cycles. For each cycle, DSS was administered until the disease activity index (DAI) reached a score of 2.0 to 2.5 (see scoring criteria below) in at least one group, at which time the 4% DSS was replaced with plain drinking water. The first cycle of DSS administration was followed by 14 days of plain drinking water. The second cycle of DSS was followed by 8 to 9 days of plain drinking water, at which time the mice were sacrificed.

[0297] Oligonucleotide was administered subcutaneously (s.c.) for four consecutive days starting on the second day of the first cycle, and then every other day thereafter at doses of 0.25 mg/kg, 2.5 mg/kg, and 12.5 mg/kg; or 0.5 and 2.5 mg/kg. TNF-α mAb was administered s.c. one time at the beginning of each cycle for a total of two treatments at 30 μg/mouse.

[0298] Chronic colitis progression was determined by daily measurement of the Disease Activity Index (DAI), consisting of weight loss, stool consistency and hemoccult scores (Cooper et al., 1993, supra.). Each parameter was given a score (Table 30) and the combined score was divided by three to obtain the disease activity index (DAI). This method has been shown to correlate with the histological measures of inflammation and crypt damage.

[0299] The damage to the crypts and extent of recovery were determined by histological analysis of the proximal and distal sections of the colon based on the crypt grade and percent involvement in each section. Crypt grades were scored as Grade 0=intact crypt; Grade 1=loss of ⅓ crypt; Grade 2=loss of ⅔ of crypt; Grade 3=loss of entire crypt w/intact epithelium; and Grade 4=loss of entire crypt w/loss of epithelium (ulceration). Percent involvement was scored as 1 =1-25%; 2=26-50%; 3=51-75%; and 4=76-100%. Total crypt score is the combined scores of the distal and proximal colon sections. The inflammation score is the product of the grade of inflammation and the extent of involvement, where Grade 0=normal; Grade 1=mild; Grade 2=moderate; Grade 3=Severe; and Percent Involvement 1=1-25%; 2=26-50%; 3=51-75%; 4=76-100%.

[0300] Total RNA was isolated from a 1 mm full length colon strip from each animal using the RNeasy Mini Kit (Qiagen, Valencia Calif.). Mouse TNFα and G3PDH mRNA levels were determined by standard northern blot procedures. TNFα probe signals were normalized to G3PDH probe signal.

[0301] Differences between treatment groups were evaluated by analysis of variance (ANOVA). If a statistically significant difference was detected by ANOVA then the Dunnett's test was applied (SAS Institute Inc., Cary N.C.).

[0302] Previous studies have examined the distribution of the “first-generation” phosphorothioate oligodeoxynucleotides in colon tissue of normal and DSS-treated mice, and demonstrated localization of oligonucleotide in both the lamina propia and the epithelial cells of the mucosal layer (Bennett, 1997, supra.). In this case, differences were observed between the two groups of mice with respect to degree of tissue accumulation as well as relative distribution between the lamina propia and epithelial cells. Disruption of the epithelial mucosa layer and influx of immune cells into the lamina propia in the DSS-treated mice coincided with increased accumulation of the oligonucleotide in the tissue, particularly in the epithelial layer.

[0303] To obtain information on the localization of a 2′-O-(2-methoxyethyl) modified (2′-MOE) phosphorothioate oligodeoxynucleotide a similar experiment was performed using immunohistochemical staining techniques, instead of autoradiographic or fluorescent techniques, to detect the oligonucleotide (Butler et al., 1997, supra.) in the colon tissue. Immunohistochemical staining allows for direct detection of the oligonucleotide without further labeling steps during oligonucleotide synthesis. The previously identified anti-oligonucleotide monoclonal antibody, 2E1, was utilized for this purpose (Butler, 1997, supra.). Cumulative studies have shown that the strength of the signal obtained from histological staining of an oligonucleotide with the 2E1 antibody is dependent on the oligonucleotide sequence. In this respect, the staining signal for ISIS 25302 proved to be modest. For this reason we utilized ISIS 13920, a 2′-MOE modified phosphorothioate oligodeoxynucleotide with enhanced histological staining properties, to evaluate the distribution of this type of oligonucleotide in colon tissue of normal and DSS-treated mice. A similar distribution and accumulation profile was observed with the “second-generation” 2′-MOE modified phosphorothioate oligodeoxynucleotide, as had previously been observed for a rhodamine labeled “first-generation” phosphorothioate oligodeoxynucleotide (Bennett, 1997, supra. ) . Enhanced staining by the anti-oligonucleotide antibody, 2E1, was observed in the colon tissue of DSS-treated mice, in comparison to the normal mice.

[0304] Mice treated with ISIS 25302 every other day at a dose of 1 mg/kg in the acute model of DSS-induced colitis showed a 44% reduction in the disease activity index (DAI) relative to the saline treated control group (1.4±0.2 vs 2.6±0.2; FIG. 5A). In comparison, mice treated one time with 25 micrograms of the anti-TNF-α mAb, at the commencement of DSS-induction, showed a 57% reduction in the DAI. In both cases, the reduction in DAI was significant (p<0.05) in comparison to the saline treated group. In contrast to the other two treatments, mice treated with 50 micrograms of antibody showed no improvement in the DAI. Improvement in the DAI correlated with an increase in colon length (FIG. 5B). The mean colon length of the saline treated DSS-induced mice was 57% the length of normal mice (see also Okayasu, 1990, supra.), whereas those of the ISIS 25302 and anti-TNF-α antibody (25 μg) treated mice were 76% and 79% respectively. The mean colon lengths of each of the two anti-TNF-α treated groups were significantly different from both the saline treated DSS-induced mice and normal mice (p<0.05).

[0305] The effect of ISIS 25302 on the development of acute colitis was dose and sequence dependent (FIG. 6A-6B). A reduction of the clinical symptoms of DSS-induced colitis, as measured by the DAI, was observed in mice treated with 0.04 (60%), 0.2 (60%), and 1 mg/kg (80%) of ISIS 25302 relative to saline treated control mice. Mice treated with the eight base mismatch control oligonucleotide, ISIS 30782, showed no reduction in the DAI in comparison to the saline treated group. The reduction in DAI in mice treated with ISIS 25302 at 0.04, 0.2, and 1.0 mg/kg was statistically significant in comparison to mice treated with the eight base mismatch control oligonucleotide at 1.0 mg/kg (p<0.05). A statistically significant difference was also observed between the 1.0 mg/kg ISIS 25302 group and the saline treated group. Treatment of the mice with ISIS 25302 at the higher dose of 5 mg/kg, yielded no improvement in the DAI; as previously observed in mice treated with 50 micrograms of the anti-TNF-α mAb (described below). A partial loss of efficacy was also observed in the acute DSS-induced colitis model with the anti-ICAM-1 oligonucleotide, ISIS 3082, at a dose of 5 mg/kg (Bennett, 1997, supra.). In the ICAM-1 study mice were administered oligonucleotide once a day for five consecutive days, instead of every other day for a total of five injections. Loss of efficacy, in all applications, may have resulted from an excessive accumulation of the oligonucleotide (or antibody) in the inflamed tissue, which in turn had an adverse effect on the animals (immune) response to intestinal injury by DSS.

[0306] ISIS 25302 was also tested for efficacy in the chronic model of DSS-induced mouse colitis. In this model, DSS was administered a second time, fourteen days after the first period of DSS administration. Animals were treated with ISIS 25302 prior to establishment of disease, starting on Day 2 of the first cycle of DSS administration. A dose-dependent reduction in the clinical signs of chronic colitis was observed in the mice treated with ISIS 25302 (FIG. 7A). For example, a 49% reduction (0.88±0.17) in the disease activity index (DAI) was observed in mice treated at the lowest dose of 0.25 mg/kg of ISIS 25302, in comparison to the saline treated control group (1.7±0.3) at the end of the second cycle (Day 10, FIG. 7B). A greater reduction in the DAI, 86 to 87%, was observed in mice treated at the higher doses of 2.5 and 12.5 mg/kg of ISIS 25302 (0.22±0.11 and 0.27±0.11, respectively). In comparison, animals treated with the anti-TNF-α mAb showed a 61% reduction in DAI (0.67±0.14). At this time the reductions in DAI scores were statistically significant (p<0.05) in mice treated with either the anti-TNF-α mAb or ISIS 25302, at all three doses, in comparison to the vehicle group. Mice that showed an improvement in DAI also showed a reduction in inflammatory infiltrates and crypt damage at the histological level as compared to the untreated and vehicle groups (FIG. 8A-B). For example, mice treated with ISIS 25302 at 2.5 and 12.5 mg/kg demonstrated a 43% and 52% reduction in total inflammatory infiltrates (respectively), and a 43% and 48% reduction in total crypt damage relative to vehicle (FIG. 8A). The proximal region of the colon was more responsive to treatment by ISIS 25302, than the distal region (FIG. 8B). However, the severity of the disease was greater in the distal region of the colon.

[0307] Although not statistically significant, a thirty percent reduction in target TNF-α mRNA levels was observed in the colon tissue of mice treated at the higher doses of 2.5 and 12.5 mg/kg ISIS 25302 (FIG. 9). The TNFα mRNA levels in colons from mice treated at the lower dose of 0.25 mg/kg of ISIS 25302 were not reduced in comparison to the vehicle group. The reduced levels of TNFα mRNA observed for mice treated with the two higher doses of ISIS 25302 supports the dose-dependent response observed in the clinic, as measured by the DAI.

[0308] The anti-mTNF-α oligoncucleotide, ISIS 25302, showed dose and sequence-specific efficacy in both the acute and chronic indications of DSS-induced colitis. ISIS 25302 treatment was also comparable in effect to treatment with an anti-TNF mAb in both indications. The reduction in the clinical symptoms observed in DSS-induced mice treated with ISIS 25302 correlated with a reduction of inflammatory infiltrates and crypt damage. Target TNF-α mRNA levels were also reduced in colon tissue derived from DSS-induced animals treated with ISIS 25302, relative to vehicle controls. The efficacy of ISIS 25302 in both the acute and chronic models of DSS-induced mouse colitis indicates that an antisense oligonucleotide which targets TNFα mRNA represents a novel approach for treatment of human inflammatory bowel disease.

Example 16 Effect of TNF-α Antisense Oligonucleotides in a Murine Model for Crohn's Disease

[0309] C3H/HeJ, SJL/JK and IL10−/− mice are used in a TNBS (2,4,5,-trinitrobenzene sulfonic acid) induced colitis model for Crohn's disease (Neurath, M. F., et al., J. Exp. Med., 1995, 182, 1281-1290). Mice between the ages of 6 weeks and 3 months are used to assess the activity of TNFα antisense oligonucleotides.

[0310] C3H/HeJ, SJL/JK and IL10−/− mice are fasted overnight prior to administration of TNBS. A thin, flexible polyethylene tube is slowly inserted into the colon of the mice so that the tip rests approximately 4 cm proximal to the anus. 0.5 mg of the TNBS in 50% ethanol is slowly injected from the catheter fitted onto a 1 ml syringe. Animals are held inverted in a vertical position for approximately 30 seconds. TNFα antisense oligonucleotides are administered either at the first sign of symptoms or simultaneously with induction of disease. Animals, in most cases, are dosed every day. Administration is by i.v., i.p., s.q., minipumps or intracolonic injection. Experimental tissues are collected at the end of the treatment regimen for histochemical evaluation.

Example 17 Effect of TNF-α Antisense Oligonucleotides in a Murine Model for Multiple Sclerosis

[0311] Experimental autoimmune encephalomyelitis (EAE) is a commonly accepted murine model for multiple sclerosis (Myers, K. J., et al., J. Neuroimmunol., 1992, 41, 1-8). SJL/H, PL/J, (SJLxPL/J)Fl, (SJLxBalb/c)F1 and Balb/c female mice between the ages of 6 and 12 weeks are used to test the activity of TNFα antisense oligonucleotides.

[0312] The mice are immunized in the two rear foot pads and base of the tail with an emulsion consisting of encephalitogenic protein or peptide (according to Myers, K. J., et al., J. of Immunol., 1993, 151, 2252-2260) in Complete Freund's Adjuvant supplemented with heat killed Mycobacterium tuberculosis. Two days later, the mice receive an intravenous injection of 500 ng Bordatella pertussis toxin and additional adjuvant.

[0313] Alternatively, the disease may also be induced by the adoptive transfer of T-cells. T-cells are obtained from the draining of the lymph nodes of mice immunized with encephalitogenic protein or peptide in CFA. The T cells are grown in tissue culture for several days and then injected intravenously into naive syngeneic recipients.

[0314] Mice are monitored and scored daily on a 0-5 scale for signals of the disease, including loss of tail muscle tone, wobbly gait, and various degrees of paralysis.

Example 18 Effect of TNFα Antisense Oligonucleotides in a Murine Model for Pancreatitis

[0315] Swiss Webster, C57BL/56, C57BL/6 lpr and gld male mice are used in an experimental pancreatitis model (Niederau, C., et al., Gastroenterology, 1985, 88, 1192-1204). Mice between the ages of 4 and 10 weeks are used to assess the activity of TNF-α antisense oligonucleotides.

[0316] Caerulin (5-200 μg/kg) is administered i.p. every hour for one to six hours. At varying time intervals, the mice are given i.p. injection of avertin and bled by cardiac puncture. The pancreas and spleen are evaluated for histopathology and increased levels of IL-1β, IL-6, and TNF-α. The blood is analyzed for increased levels of serum amylase and lipase. TNF-α antisense oligonucleotides are administered by intraperitoneal injection at 4 hours pre-caerulin injections.

Example 19 Effect of TNFα Antisense Oligonucleotides in a Murine Model for Hepatitis

[0317] Concanavalin A-induced hepatitis is used as a murine model for hepatitis (Mizuhara, H., et al., J. Exp. Med., 1994, 179, 1529-1537). It has been shown that this type of liver injury is mediated by Fas (Seino, K., et al., Gastroenterology 1997, 113, 1315-1322). Certain types of viral hepatitis, including Hepatitis C, are also mediated by Fas (J. Gastroenterology and Hepatology, 1997, 12, S223-S226). Female Balb/c and C57BL/6 mice between the ages of 6 weeks and 3 months are used to assess the activity of TNF-α antisense oligonucleotides.

[0318] Mice are intravenously injected with oligonucleotide. The pretreated mice are then intravenously injected with 0.3 mg concanavalin A (Con A) to induce liver injury. Within 24 hours following Con A injection, the livers are removed from the animals and analyzed for cell death (apoptosis) by in vitro methods. In some experiments, blood is collected from the retro-orbital vein.

Example 20 Effect of Antisense Oligonucleotide Targeted to TNF-α on Survival in Murine Heterotopic Heart Transplant Model

[0319] To determine the therapeutic effects of TNFα antisense oligonucleotides in preventing allograft rejection, murine TNF-α-specific oligonucleotides are tested for activity in a murine vascularized heterotopic heart transplant model. Hearts from Balb/c mice are transplanted into the abdominal cavity of C3H mice as primary vascularized grafts essentially as described by Isobe et al., Circulation 1991, 84, 1246-1255. Oligonucleotide is administered by continuous intravenous administration via a 7-day Alzet pump. The mean survival time for untreated mice is usually approximately 9-10 days. Treatment of the mice for 7 days with TNF-α antisense oligonucleotides is expected to increase the mean survival time.

Example 21 Optimization of Human TNF-α Antisense Oligonucleotide

[0320] Additional antisense oligonucleotides targeted to intron 1 of human TNF-α were designed. These are shown in Table 31. Oligonucleotides are screened by RT-PCR as described in Example 5 hereinabove. TABLE 31 Nucleotide Sequences of Human TNF-α Intron 1 Antisense Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′ -> 3′) NO: ORDINATES² REGION 100181 AGTGTCTTCTGTGTGCCAGA 144 1409-1428 intron 1 100201 AGTGTCTTCTGTGTGCCAGA ″ ″ intron 1 100230 AGTGTCTTCTGTGTGCCAGA ″ ″ intron 1 100250 AGTGTCTTCTGTGTGCCAGA ″ ″ intron 1 100182 GTGTCTTCTGTGTGCCAGAC 145 1408-1427 intron 1 100202 GTGTCTTCTGTGTGCCAGAC ″ ″ intron 1 100231 GTGTCTTCTGTGTGCCAGAC ″ ″ intron 1 100251 GTGTCTTCTGTGTGCCAGAC ″ ″ intron 1 100183 TGTCTTCTGTGTGCCAGACA 146 1407-1426 intron 1 100203 TGTCTTCTGTGTGCCAGACA ″ ″ intron 1 100232 TGTCTTCTGTGTGCCAGACA ″ ″ intron 1 100252 TGTCTTCTGTGTGCCAGACA ″ ″ intron 1 100184 GTCTTCTGTGTGCCAGACAC 147 1406-1425 intron 1 100204 GTCTTCTGTGTGCCAGACAC ″ ″ intron 1 100233 GTCTTCTGTGTGCCAGACAC ″ ″ intron 1 100253 GTCTTCTGTGTGCCAGACAC ″ ″ intron 1 100185 TCTTCTGTGTGCCAGACACC 148 1405-1424 intron 1 100205 TCTTCTGTGTGCCAGACACC ″ ″ intron 1 100234 TCTTCTGTGTGCCAGACACC ″ ″ intron 1 100254 TCTTCTGTGTGCCAGACACC ″ ″ intron 1 100186 CTTCTGTGTGCCAGACACCC 149 1404-1423 intron 1 100206 CTTCTGTGTGCCAGACACCC ″ ″ intron 1 100235 CTTCTGTGTGCCAGACACCC ″ ″ intron 1 100255 CTTCTGTGTGCCAGACACCC ″ ″ intron 1 100187 TTCTGTGTGCCAGACACCCT 150 1403-1422 intron 1 100207 TTCTGTGTGCCAGACACCCT ″ ″ intron 1 100236 TTCTGTGTGCCAGACACCCT ″ ″ intron 1 100256 TTCTGTGTGCCAGACACCCT ″ ″ intron 1 100188 TCTGTGTGCCAGACACCCTA 151 1402-1421 intron 1 100208 TCTGTGTGCCAGACACCCTA ″ ″ intron 1 100237 TCTGTGTGCCAGACACCCTA ″ ″ intron 1 100257 TCTGTGTGCCAGACACCCTA ″ ″ intron 1 100189 CTGTGTGCCAGACACCCTAT 152 1401-1420 intron 1 100209 CTGTGTGCCAGACACCCTAT ″ ″ intron 1 100238 CTGTGTGCCAGACACCCTAT ″ ″ intron 1 100258 CTGTGTGCCAGACACCCTAT ″ ″ intron 1 100190 TGTGTGCCAGACACCCTATC 153 1400-1419 intron 1 100210 TGTGTGCCAGACACCCTATC ″ ″ intron 1 100239 TGTGTGCCAGACACCCTATC ″ ″ intron 1 100259 TGTGTGCCAGACACCCTATC ″ ″ intron 1 100191 TGTGCCAGACACCCTATCTT 154 1398-1417 intron 1 100211 TGTGCCAGACACCCTATCTT ″ ″ intron 1 100240 TGTGCCAGACACCCTATCTT ″ ″ intron 1 100260 TGTGCCAGACACCCTATCTT ″ ″ intron 1 100192 GTGCCAGACACCCTATCTTC 155 1397-1416 intron 1 100212 GTGCCAGACACCCTATCTTC ″ ″ intron 1 100241 GTGCCAGACACCCTATCTTC ″ ″ intron 1 100261 GTGCCAGACACCCTATCTTC ″ ″ intron 1 100193 TGCCAGACACCCTATCTTCT 156 1396-1415 intron 1 100213 TGCCAGACACCCTATCTTCT ″ ″ intron 1 100242 TGCCAGACACCCTATCTTCT ″ ″ intron 1 100262 TGCCAGACACCCTATCTTCT ″ ″ intron 1 100194 GCCAGACACCCTATCTTCTT 157 1395-1414 intron 1 100214 GCCAGACACCCTATCTTCTT ″ ″ intron 1 100243 GCCAGACACCCTATCTTCTT ″ ″ intron 1 100263 GCCAGACACCCTATCTTCTT ″ ″ intron 1 100195 CCAGACACCCTATCTTCTTC 158 1394-1413 intron 1 100215 CCAGACACCCTATCTTCTTC ″ ″ intron 1 100244 CCAGACACCCTATCTTCTTC ″ ″ intron 1 100264 CCAGACACCCTATCTTCTTC ″ ″ intron 1 100196 CAGACACCCTATCTTCTTCT 159 1393-1412 intron 1 100216 CAGACACCCTATCTTCTTCT ″ ″ intron 1 100245 CAGACACCCTATCTTCTTCT ″ ″ intron 1 100265 CAGACACCCTATCTTCTTCT ″ ″ intron 1 100197 AGACACCCTATCTTCTTCTC 160 1392-1411 intron 1 100217 AGACACCCTATCTTCTTCTC ″ ″ intron 1 100246 AGACACCCTATCTTCTTCTC ″ ″ intron 1 100266 AGACACCCTATCTTCTTCTC ″ ″ intron 1 100198 GACACCCTATCTTCTTCTCT 161 1391-1410 intron 1 100218 GACACCCTATCTTCTTCTCT ″ ″ intron 1 100247 GACACCCTATCTTCTTCTCT ″ ″ intron 1 100267 GACACCCTATCTTCTTCTCT ″ ″ intron 1 100199 ACACCCTATCTTCTTCTCTC 162 1390-1409 intron 1 100219 ACACCCTATCTTCTTCTCTC ″ ″ intron 1 100248 ACACCCTATCTTCTTCTCTC ″ ″ intron 1 100268 ACACCCTATCTTCTTCTCTC ″ ″ intron 1 100200 CACCCTATCTTCTTCTCTCC 163 1389-1408 intron 1 100220 CACCCTATCTTCTTCTCTCC ″ ″ intron 1 100249 CACCCTATCTTCTTCTCTCC ″ ″ intron 1 100269 CACCCTATCTTCTTCTCTCC ″ ″ intron 1 100270 GTCTTCTGTGTGCCAGAC 164 1408-1425 intron 1 100271 TCTTCTGTGTGCCAGACA 165 1407-1424 intron 1 100272 CTTCTGTGTGCCAGACAC 166 1406-1423 intron 1 100273 TTCTGTGTGCCAGACACC 167 1405-1422 intron 1 100274 TCTGTGTGCCAGACACCC 168 1404-1421 intron 1 100275 CTGTGTGCCAGACACCCT 169 1403-1420 intron 1 100276 TGTGTGCCAGACACCCTA 170 1402-1419 intron 1 100277 GTGTGCCAGACACCCTAT 171 1401-1418 intron 1 100278 TGTGCCAGACACCCTATC 172 1400-1417 intron 1 100279 TGCCAGACACCCTATCTT 173 1398-1415 intron 1 100280 GCCAGACACCCTATCTTC 174 1397-1414 intron 1 100281 CCAGACACCCTATCTTCT 175 1396-1413 intron 1 100282 CAGACACCCTATCTTCTT 176 1395-1412 intron 1 100283 AGACACCCTATCTTCTTC 177 1394-1411 intron 1 100284 GACACCCTATCTTCTTCT 178 1393-1410 intron 1 100285 ACACCCTATCTTCTTCTC 179 1392-1409 intron 1

Example 22 Design of Antisense Oligonucleotides Targeting Human TNF-α Intron 2

[0321] Additional antisense oligonucleotides targeted to intron 2 and coding regions of human TNF-α were designed. These are shown in Table 32. Oligonucleotides are screened by RT-PCR as described in Example 5 hereinabove. TABLE 32 Nucleotide Sequences of Human TNF-α Intron 2 Antisense Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′ -> 3′) NO: ORDINATES² REGION 100549 AGAGGTTTGGAGACACTTAC 180 1635-1654 intron 2 100566 AGAGGTTTGGAGACACTTAC ″ ″ intron 2 100550 GAATTAGGAAAGAGGTTTGG 181 1645-1664 intron 2 100567 GAATTAGGAAAGAGGTTTGG ″ ″ intron 2 100551 CCCAAACCCAGAATTAGGAA 182 1655-1674 intron 2 100568 CCCAAACCCAGAATTAGGAA ″ ″ intron 2 100552 TACCCCCAAACCCAAACCCA 183 1665-1684 intron 2 100569 TACCCCCAAACCCAAACCCA ″ ″ intron 2 100553 GTACTAACCCTACCCCCAAA 184 1675-1694 intron 2 100570 GTACTAACCCTACCCCCAAA ″ ″ intron 2 100554 TTCCATACCGGTACTAACCC 185 1685-1704 intron 2 100571 TTCCATACCGGTACTAACCC ″ ″ intron 2 100555 CCCCCACTGCTTCCATACCG 186 1695-1714 intron 2 100572 CCCCCACTGCTTCCATACCG ″ ″ intron 2 100556 CTTTAAATTTCCCCCACTGC 187 1705-1724 intron 2 100573 CTTTAAATTTCCCCCACTGC ″ ″ intron 2 100557 AAGACCAAAACTTTAAATTT 188 1715-1734 intron 2 100571 AAGACCAAAACTTTAAATTT ″ ″ intron 2 100558 ATCCTCCCCCAAGAGCAAAA 189 1725-1744 intron 2 100640 ATCCTCCCCCAAGAGCAAAA ″ ″ intron 2 100559 ACCTCCATCCATCCTCCCCC 190 1735-1754 intron 2 100641 ACCTCCATCCATCCTCCCCC ″ ″ intron 2 100560 CCCTACTTTCACCTCCATCC 191 1745-1764 intron 2 100642 CCCTACTTTCACCTCCATCC ″ ″ intron 2 100561 GAAAATACCCCCCTACTTTC 192 1755-1774 intron 2 100643 GAAAATACCCCCCTACTTTC ″ ″ intron 2 100562 AAACTTCCTAGAAAATACCC 193 1765-1784 intron 2 100644 AAACTTCCTAGAAAATACCC ″ ″ intron 2 100563 TGAGACCCTTAAACTTCCTA 194 1775-1794 intron 2 100645 TGAGACCCTTAAACTTCCTA ″ ″ intron 2 100564 AAGAAAAAGCTGAGACCCTT 195 1785-1804 intron 2 100646 AAGAAAAAGCTGAGACCCTT ″ ″ intron 2 100565 GGAGAGAGAAAAGAAAAAGC 196 1795-1814 intron 2 100647 GGAGAGAGAAAAGAAAAAGC ″ ″ intron 2 100575 TGAGCCAGAAGAGGTTGAGG 197 2665-2684 coding 100576 ATTCTCTTTTTGAGCCAGAA 198 2675-2694 coding 100577 TAAGCCCCCAATTCTCTTTT 199 2685-2704 coding 100578 GTTCCGACCCTAAGCCCCCA 200 2695-2714 coding 100579 CTAAGCTTGGGTTCCGACCC 201 2705-2724 coding 100580 GCTTAAAGTTCTAAGCTTGG 202 2715-2734 coding 100581 TGGTCTTGTTGCTTAAAGTT 203 2725-2744 coding 100582 TTCGAAGTGGTGGTCTTGTT 204 2735-2754 coding 100583 AATCCCAGGTTTCGAAGTGG 205 2745-2764 coding 100584 CACATTCCTGAATCCCAGGT 206 2755-2774 coding 100585 GTGCAGGCCACACATTCCTG 207 2765-2784 coding 100586 GCACTTCACTGTGCAGGCCA 208 2775-2794 coding 100587 GTGGTTGCCAGCACTTCACT 209 2785-2804 coding 100588 TGAATTCTTAGTGGTTGCCA 210 2795-2814 coding 100589 GGCCCCAGTTTGAATTCTTA 211 2805-2824 coding 100590 GAGTTCTGGAGGCCCCAGTT 212 2815-2834 coding 100591 AGGCCCCAGTGAGTTCTGGA  32 2825-2844 coding 100592 TCAAAGCTGTAGGCCCCAGT 214 2835-2854 coding 100593 ATGTCAGGGATCAAAGCTGT 215 2845-2864 coding 100594 CAGATTCCAGATGTCAGGGA 216 2855-2874 coding 100595 CCCTGGTCTCCAGATTCCAG 217 2865-2884 coding 100596 ACCAAAGGCTCCCTGGTCTC 218 2875-2894 coding 100597 TCTGGCCAGAACCAAAGGCT 219 2885-2904 coding 100598 CCTGCAGCATTCTGGCCAGA 220 2895-2914 coding 100599 CTTCTCAAGTCCTGCAGCAT 221 2905-2924 coding 100600 TAGGTGAGGTCTTCTCAAGT 222 2915-2934 coding 100601 TGTCAATTTCTAGGTGAGGT 223 2925-2944 coding 100602 GGTCCACTTGTGTCAATTTC 224 2935-2954 coding 100603 GAAGGCCTAAGGTCCACTTG 225 2945-2964 coding 100604 CTGGAGAGAGGAAGGCCTAA 226 2955-2974 coding 100605 CTGGAAACATCTGGAGAGAG 227 2965-2984 coding 100606 TCAAGGAAGTCTGGAAACAT 228 2975-2994 coding 100607 GCTCCGTGTCTCAAGGAAGT 229 2985-3004 coding 100608 ATAAATACATTCATCTGTAA 230 3085-3104 coding 100609 GGTCTCCCAAATAAATACAT 231 3095-3114 coding 100610 AGGATACCCCGGTCTCCCAA 232 3105-3124 coding 100611 TGGGTCCCCCAGGATACCCC  35 3115-3134 coding 100612 GCTCCTACATTGGGTCCCCC 234 3125-3144 coding 100613 AGCCAAGGCAGCTCCTACAT 235 3135-3154 coding 100614 AACATGTCTGAGCCAAGGCA 236 3145-3164 coding 100615 TTTCACGGAAAACATGTCTG 237 3155-3174 coding 100616 TCAGCTCCGTTTTCACGGAA 238 3165-3184 coding 100617 AGCCTATTGTTCAGCTCCGT 239 3175-3194 coding 100618 ACATGGGAACAGCCTATTGT 240 3185-3204 coding 100619 ATCAAAAGAAGGCACAGAGG 241 3215-3234 coding 100620 GTTTAGACAACTTAATCAGA 242 3255-3274 coding 100621 AATCAGCATTGTTTAGACAA 243 3265-3284 coding 100622 TTGGTCACCAAATCAGCATT 244 3275-3294 coding 100623 TGAGTGACAGTTGGTCACCA 245 3285-3304 coding 100624 GGCTCAGCAATGAGTGACAG 246 3295-3314 coding 100625 ATTACAGACACAACTCCCCT 247 3325-3344 coding 100626 TAGTAGGGCGATTACAGACA 248 3335-3354 coding 100627 CGCCACTGAATAGTAGGGCG 249 3345-3364 coding 100628 CTTTATTTCTCGCCACTGAA 250 3355-3374 coding

[0322] Several of these oligonucleotides were chosen for dose response studies. Cells were grown and treated as described in Example 3. Results are shown in Table 33. Each oligonucleotide tested showed a dose response curve with maximum inhibition greater than 75%. TABLE 33 Dose Response of PMA-Induced neoHK Cells to TNF-α Antisense Oligonucleotides (ASOs) ASO Gene % protein % protein ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — — 100% — 100235 149 intron 1  75 nM 77% 23% ″ ″ ″ 150 nM 25% 75% ″ ″ ″ 300 nM 6% 94% 100243 157 intron 1  75 nM 68% 32% ″ ″ ″ 150 nM 15% 85% ″ ″ ″ 300 nM 6% 94% 100263 157 intron 1  75 nM 79% 21% ″ ″ ″ 150 nM 30% 70% ″ ″ ″ 300 nM 23% 77%

Example 23 Optimization of Human TNF-α Antisense Oligonucleotide Chemistry

[0323] Analogs of oligonucleotides 21820 (SEQ ID NO. 66) and 21823 (SEQ ID NO. 69) were designed and synthesized to find an optimum gap size. The sequences and chemistries are shown in Table 34.

[0324] Dose response experiments were performed as described in Example 3. Results are shown in Table 35. TABLE 34 Nucleotide Sequences of TNF-α Chimeric Backbone (deoxy gapped) Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′ -> 3′) NO: ORDINATES² REGION 21820 ATATTTCCCGCTCTTTCTGT 66 1339-1358 intron 1 28086 ATATTTCCCGCTCTTTCTGT ″ ″ ″ 28087 ATATTTCCCGCTCTTTCTGT ″ ″ ″ 21823 GTGTGCCAGACACCCTATCT 69 1399-1418 intron 1 28088 GTGTGCCAGACACCCTATCT ″ ″ ″ 28089 GTGTGCCAGACACCCTATCT ″ ″ ″

[0325] TABLE 35 Dose Response of 20 Hour PMA-Induced neoHK Cells to TNF-α Chimeric (deoxy gapped) Antisense Oligonucleotides (ASOs) ASO Gene % protein % protein ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — —  100% — 13393 49 control  75 nM 150.0%  — ″ ″ ″ 150 nM 135.0%  — ″ ″ ″ 300 nM 90.0% 10.0% 21820 66 intron 1  75 nM 65.0% 35.0% ″ ″ ″ 150 nM 28.0% 72.0% ″ ″ ″ 300 nM  9.7% 90.3% 28086 66 intron 1  75 nM 110.0%  — ″ ″ ″ 150 nM 83.0% 17.0% ″ ″ ″ 300 nM 61.0% 39.0% 28087 66 intron 1  75 nM 127.0%  — ″ ″ ″ 150 nM 143.0%  — ″ ″ ″ 300 nM 147.0%  — 21823 69 intron 1  75 nM 35.0% 65.0% ″ ″ ″ 150 nM 30.0% 70.0% ″ ″ ″ 300 nM  6.4% 93.6% 28088 69 intron 1  75 nM 56.0% 44.0% ″ ″ ″ 150 nM 26.0% 74.0% ″ ″ ″ 300 nM 11.0% 89.0% 28089 69 intron 1  75 nM 76.0% 24.0% ″ ″ ″ 150 nM 53.0% 47.0% ″ ″ ″ 1300 nM  23.0% 77.0%

Example 24 Screening of Additional TNFα Chimeric (Deoxy Gapped) Antisense Oligonucleotides

[0326] Additional oligonucleotides targeting the major regions of TNF-α were synthesized. Oligonucleotides were synthesized as uniformly phosphorothioate chimeric oligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central region of ten deoxynucleotides. Oligonucleotide sequences are shown in Table 36.

[0327] Oligonucleotides were screened as described in Example 5. Results are shown in Table 37. TABLE 36 Nucleotide Sequence of Additional Human TNF-α Chimeric (deoxy gapped) Antisense Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES² REGION 104649 CTGAGGGAGCGTCTGCTGGC 251 0616-0635 5′-UTR 104650 CCTTGCTGAGGGAGCGTCTG 252 0621-0640 5′-UTR 104651 CTGGTCCTCTGCTGTCCTTG 253 0636-0655 5′-UTR 104652 CCTCTGCTGTCCTTGCTGAG 254 0631-0650 5′-UTR 104653 TTCTCTCCCTCTTAGCTGGT 255 0651-0670 5′-UTR 104654 TCCCTCTTAGCTGGTCCTCT 256 0646-0665 5′-UTR 104655 TCTGAGGGTTGTTTTCAGGG 257 0686-0705 5′-UTR 104656 CTGTAGTTGCTTCTCTCCCT 258 0661-0680 5′-UTR 104657 ACCTGCCTGGCAGCTTGTCA 259 0718-0737 5′-UTR 104658 GGATGTGGCGTCTGAGGGTT 260 0696-0715 5′-UTR 104659 TGTGAGAGGAAGAGAACCTG 261 0733-0752 5′-UTR 104660 GAGGAAGAGAACCTGCCTGG 262 0728-0747 5′-UTR 104661 AGCCGTGGGTCAGTATGTGA 263 0748-0767 5′-UTR 104662 TGGGTCAGTATGTGAGAGGA 264 0743-0762 5′-UTR 104663 GAGAGGGTGAAGCCGTGGGT 265 0758-0777 5′-UTR 104664 TCATGGTGTCCTTTCCAGGG 266 0780-0799 AUG 104665 CTTTCAGTGCTCATGGTGTC 267 0790-0809 AUG 104666 TCATGCTTTCAGTGCTCATG 268 0795-0814 AUG 104667 ACGTCCCGGATCATGCTTTC 269 0805-0824 coding 104668 GCTCCACGTCCCGGATCATG 270 0810-0829 coding 104669 TCCTCGGCCAGCTCCACGTC 271 0820-0839 coding 104670 GCGCCTCCTCGGCCAGCTCC 272 0825-0844 coding 104671 AGGAACAAGCACCGCCTGGA 273 0874-0893 coding 104672 CAAGCACCGCCTGGAGCCCT 274 0869-0888 coding 104673 AAGGAGAAGAGGCTGAGGAA 275 0889-0908 coding 104674 GAAGAGGCTGAGGAACAAGC 276 0884-0903 coding 104675 CCTGCCACGATCAGGAAGGA 277 0904-0923 coding 104676 CACGATCAGGAAGGAGAAGA 278 0899-0918 coding 104677 AAGAGCGTGGTGGCGCCTGC 279 0919-0938 coding 104678 CGTGGTGGCGCCTGCCACGA 280 0914-0933 coding 104679 AAGTGCAGCAGGCAGAAGAG 281 0934-0953 coding 104680 CAGCAGGCAGAAGAGCGTGG 282 0929-0948 coding 104681 GATCACTCCAAAGTGCAGCA 283 0944-0963 coding 104682 GGGCCGATCACTCCAAAGTG 284 0949-0968 coding 104683 GGGCCAGAGGGCTGATTAGA 285 1606-1625 coding 104684 AGAGGGCTGATTAGAGAGAG 286 1601-1620 coding 104685 GCTACAGGCTTGTCACTCGG 287 1839-1858 coding 104686 CTGACTGCCTGGGCCAGAGG 288 1616-1635 E2/I2³ 104687 TACAACATGGGCTACAGGCT 289 1849-1868 coding 104688 AGCCACTGGAGCTGCCCCTC 290 2185-2204 coding 104689 CTGGAGCTGCCCCTCAGCTT 291 2180-2199 coding 104690 TTGGCCCGGCGGTTCAGCCA 292 2200-2219 coding 104691 TTGGCCAGGAGGGCATTGGC 293 2215-2234 coding 104692 CCGGCGGTTCAGCCACTGGA 294 2195-2214 coding 104693 CTCAGCTCCACGCCATTGGC 295 2230-2249 coding 104694 CAGGAGGGCATTGGCCCGGC 296 2210-2229 coding 104695 CTCCACGCCATTGGCCAGGA 297 2225-2244 coding 104696 ACCAGCTGGTTATCTCTCAG 298 2245-2264 coding 104697 CTGGTTATCTCTCAGCTCCA 299 2240-2259 coding 104698 CCCTCTGATGGCACCACCAG 300 2260-2279 coding 104699 TGATGGCACCACCAGCTGGT 301 2255-2274 coding 104700 TAGATGAGGTACAGGCCCTC 302 2275-2294 coding 104701 AAGAGGACCTGGGAGTAGAT 303 2290-2309 coding 104702 GAGGTACAGGCCCTCTGATG 304 2270-2289 coding 104703 CAGCCTTGGCCCTTGAAGAG 305 2305-2324 coding 104704 GACCTGGGAGTAGATGAGGT 306 2285-2304 coding 104705 TTGGCCCTTGAAGAGGACCT 307 2300-2319 coding 104706 TGGTGTGGGTGAGGAGCACA 308 2337-2356 coding 104707 CGGCGATGCGGCTGATGGTG 309 2352-2371 coding 104708 TGGGTGAGGAGCACATGGGT 310 2332-2351 coding 104709 TGGTCTGGTAGGAGACGGCG 311 2367-2386 coding 104710 ATGCGGCTGATGGTGTGGGT 312 2347-2366 coding 104711 AGAGGAGGTTGACCTTGGTC 313 2382-2401 coding 104712 TGGTAGGAGACGGCGATGCG 314 2362-2381 coding 104713 AGGTTGACCTTGGTCTGGTA 315 2377-2396 coding 104714 GGCTCTTGATGGCAGAGAGG 316 2397-2416 coding 104715 TCATACCAGGGCTTGGCCTC 317 2446-2465 coding 104716 TTGATGGCAGAGAGGAGGTT 318 2392-2411 coding 104717 CCCAGATAGATGGGCTCATA  93 2461-2480 coding 104718 CCAGGGCTTGGCCTCAGCCC  94 2441-2460 coding 104719 AGCTGGAAGACCCCTCCCAG 319 2476-2495 coding 104720 ATAGATGGGCTCATACCAGG 320 2456-2475 coding 104721 CGGTCACCCTTCTCCAGCTG 321 2491-2510 coding 104722 GAAGACCCCTCCCAGATAGA 322 2471-2490 coding 104723 ATCTCAGCGCTGAGTCGGTC  26 2506-2525 coding 104724 ACCCTTCTCCAGCTGGAAGA 323 2486-2505 coding 104725 TAGTCGGGCCGATTGATCTC  90 2521-2540 coding 104726 AGCGCTGAGTCGGTCACCCT  91 2501-2520 coding 104727 TCGGCAAAGTCGAGATAGTC 324 2536-2554 coding 104728 GGGCCGATTGATCTCAGCGC 325 2516-2535 coding 104729 TAGACCTGCCCAGACTCGGC 326 2551-2570 coding 104730 AAAGTCGAGATAGTCGGGCC 327 2531-2550 coding 104731 GCAATGATCCCAAAGTAGAC 328 2566-2585 coding 104732 CTGCCCAGACTCGGCAAAGT 329 2546-2565 coding 104733 CGTCCTCCTCACAGGGCAAT 330 2581-2600 stop 104734 GATCCCAAAGTAGACCTGCC  88 2561-2580 coding 104735 GGAAGGTTGGATGTTCGTCC 331 2596-2615 3′-UTR 104736 TCCTCACAGGGCAATGATCC 332 2576-2595 stop 104737 GTTGAGGGTGTCTGAAGGAG 333 2652-2671 3′-UTR 104738 GTTGGATGTTCGTCCTCCTC 334 2591-2610 stop 104739 TTTGAGCCAGAAGAGGTTGA 335 2667-2686 3′-UTR 104740 GAGGCGTTTGGGAAGGTTGG 336 2606-2625 3′-UTR 104741 GCCCCCAATTCTCTTTTTGA 337 2682-2701 3′-UTR 104742 GCCAGAAGAGGTTGAGGGTG 338 2662-2681 3′-UTR 104743 GGGTTCCGACCCTAAGCCCC 339 2697-2716 3′-UTR 104744 CAATTCTCTTTTTGAGCCAG 340 2677-2696 3′-UTR 104745 TAAAGTTCTAAGCTTGGGTT 341 2712-2731 3′-UTR 104746 CCGACCCTAAGCCCCCAATT 342 2692-2711 3′-UTR 104747 GGTGGTCTTGTTGCTTAAAG 343 2727-2746 3′-UTR 104748 TTCTAAGCTTGGGTTCCGAC 344 2707-2726 3′-UTR 104749 CCCAGGTTTCGAAGTGGTGG 345 2742-2761 3′-UTR 104750 TCTTGTTGCTTAAAGTTCTA 346 2722-2741 3′-UTR 104751 CACACATTCCTGAATCCCAG 347 2757-2776 3′-UTR 104752 GTTTCGAAGTGGTGGTCTTG 348 2737-2756 3′-UTR 104753 CTTCACTGTGCAGGCCACAC 349 2772-2791 3′-UTR 104754 ATTCCTGAATCCCAGGTTTC 350 2752-2771 3′-UTR 104755 TAGTGGTTGCCAGCACTTCA 351 2787-2806 3′-UTR 104756 CCCAGTTTGAATTCTTAGTG 352 2802-2821 3′-UTR 104757 CTGTGCAGGCCACACATTCC 353 2767-2786 3′-UTR 104758 GTGAGTTCTGGAGGCCCCAG 354 2817-2836 3′-UTR 104759 GTTGCCAGCACTTCACTGTG 355 2782-2801 3′-UTR 104760 TTTGAATTCTTAGTGGTTGC 356 2797-2816 3′-UTR 104761 AAGCTGTAGGCCCCAGTGAG 357 2832-2851 3′-UTR 104762 TTCTGGAGGCCCCAGTTTGA 358 2812-2831 3′-UTR 104763 AGATGTCAGGGATCAAAGCT 359 2847-2866 3′-UTR 104764 TGGTCTCCAGATTCCAGATG 360 2862-2881 3′-UTR 104765 GTAGGCCCCAGTGAGTTCTG 361 2827-2846 3′-UTR 104766 GAACCAAAGGCTCCCTGGTC 362 2877-2896 3′-UTR 104767 TCAGGGATCAAAGCTGTAGG 363 2842-2861 3′-UTR 104768 TCCAGATTCCAGATGTCAGG 364 2857-2876 3′-UTR 104769 GCAGCATTCTGGCCAGAACC 365 2892-2911 3′-UTR 104770 GTCTTCTCAAGTCCTGCAGC 366 2907-2926 3′-UTR 104771 AAAGGCTCCCTGGTCTCCAG 367 2872-2891 3′-UTR 104772 CAATTTCTAGGTGAGGTCTT 368 2922-2941 3′-UTR 104773 ATTCTGGCCAGAACCAAAGG 369 2887-2906 3′-UTR 104774 CTCAAGTCCTGCAGCbATTCT  34 2902-2921 3′-UTR 104775 AAGGTCCACTTGTGTCAATT 370 2937-2956 3′-UTR 104776 GAGAGAGGAAGGCCTAAGGT 371 2952-2971 3′-UTR 104777 TCTAGGTGAGGTCTTCTCAA 372 2917-2936 3′-UTR 104778 CCACTTGTGTCAATTTCTAG 373 2932-2951 3′-UTR 104779 GTCTGGAAACATCTGGAGAG 374 2967-2986 3′-UTR 104780 CCGTGTCTCAAGGAAGTCTG 375 2982-3001 3′-UTR 104781 AGGAAGGCCTAAGGTCCACT 376 2947-2966 3′-UTR 104782 GAGGGAGCTGGCTCCATGGG 377 3014-3033 3′-UTR 104783 GAAACATCTGGAGAGAGGAA 378 2962-2981 3′-UTR 104784 GTGCAAACATAAATAGAGGG 379 3029-3048 3′-UTR 104785 TCTCAAGGAAGTCTGGAAAC 380 2977-2996 3′-UTR 104786 AATAAATAATCACAAGTGCA 381 3044-3063 3′-UTR 104787 GGGCTGGGCTCCGTGTCTCA 382 2992-3011 3′-UTR 104788 TACCCCGGTCTCCCAAATAA 383 3101-3120 3′-UTR 104789 AACATAAATAGAGGGAGCTG 384 3024-3043 3′-UTR 104790 TTGGGTCCCCCAGGATACCC 385 3116-3135 3′-UTR 104791 ATAATCACAAGTGCAAACAT 386 3039-3058 3′-UTR 104792 AAGGCAGCTCCTACATTGGG 387 3131-3150 3′-UTR 104793 CGGTCTCCCAAATAAATACA 388 3096-3115 3′-UTR 104794 AAACATGTCTGAGCCAAGGC 389 3146-3165 3′-UTR 104795 TCCCCCAGGATACCCCGGTC 390 3111-3130 3′-UTR 104796 AGCTCCTACATTGGGTCCCC 391 3126-3145 3′-UTR 104797 CTCCGTTTTCACGGAAAACA  37 3161-3180 3′-UTR 104798 TGTCTGAGCCAAGGCAGCTC 392 3141-3160 3′-UTR 104799 CAGCCTATTGTTCAGCTCCG 393 3176-3195 3′-UTR 104800 AGAAGGCACAGAGGCCAGGG 394 3209-3228 3′-UTR 104801 TTTTCACGGAAAACATGTCT 395 3156-3175 3′-UTR 104802 TATTGTTCAGCTCCGTTTTC 396 3171-3190 3′-UTR 104803 AAAAACATAATCAAAAGAAG 397 3224-3243 3′-UTR 104804 CAGATAAATATTTTAAAAAA 398 3239-3258 3′-UTR 104805 TACATGGGAACAGCCTATTG 399 3186-3205 3′-UTR 104806 TTTAGACAACTTAATCAGAT 400 3254-3273 3′-UTR 104807 CATAATCAAAAGAAGGCACA 401 3219-3238 3′-UTR 104808 ACCAAATCAGCATTGTTTAG 402 3269-3288 3′-UTR 104809 AAATATTTTAAAAAACATAA 403 3234-3253 3′-UTR 104810 GAGTGACAGTTGGTCACCAA 404 3284-3303 3′-UTR 104811 ACAACTTAATCAGATAAATA 405 3249-3268 3′-UTR 104812 CAGAGGCTCAGCAATGAGTG 406 3299-3318 3′-UTR 104813 ATCAGCATTGTTTAGACAAC 407 3264-3283 3′-UTR 104814 AGGGCGATTACAGACACAAC 408 3331-3350 3′-UTR 104815 ACAGTTGGTCACCAAATCAG 409 3279-3298 3′-UTR 104816 TCGCCACTGAATAGTAGGGC 410 3346-3365 3′-UTR 104817 GCTCAGCAATGAGTGACAGT 411 3294-3313 3′-UTR 104818 AGCAAACTTTATTTCTCGCC 412 3361-3380 3′-UTR 104819 GATTACAGACACAACTCCCC 413 3326-3345 3′-UTR 104820 ACTGAATAGTAGGGCGATTA 414 3341-3360 3′-UTR 104821 ACTTTATTTCTCGCCACTGA 415 3356-3375 3′-UTR 104822 GCTGTCCTTGCTGAGGGAGC 416 0626-0645 5′-UTR 104823 CTTAGCTGGTCCTCTGCTGT 417 0641-0660 5′-UTR 104824 GTTGCTTCTCTCCCTCTTAG 418 0656-0675 5′-UTR 104825 TGGCGTCTGAGGGTTGTTTT 419 0691-0710 5′-UTR 104826 AGAGAACCTGCCTGGCAGCT 420 0723-0742 5′-UTR 104827 CAGTATGTGAGAGGAAGAGA 421 0738-0757 5′-UTR 104828 GGTGAAGCCGTGGGTCAGTA 422 0753-0772 5′-UTR 104829 AGTGCTCATGGTGTCCTTTC 423 0785-0804 AUG 104830 CCGGATCATGCTTTCAGTGC 424 0800-0819 coding 104831 GGCCAGCTCCACGTCCCGGA 425 0815-0834 coding 104832 GGCCCCCCTGTCTTCTTGGG 426 0847-0866 coding 104833 GGCTGAGGAACAAGCACCGC 427 0879-0898 coding 104834 TCAGGAAGGAGAAGAGGCTG 428 0894-0913 coding 104835 TGGCGCCTGCCACGATCAGG 429 0909-0918 coding 104836 GGCAGAAGAGCGTGGTGGCG 430 0924-0943 coding 104837 CTCCAAAGTGCAGCAGGCAG 431 0939-0958 coding 104838 GCTGATTAGAGAGAGGTCCC 432 1596-1615 coding 104839 TGCCTGGGCCAGAGGGCTGA 433 1611-1630 coding 104840 GCTGCCCCTCAGCTTGAGGG 434 2175-2194 coding 104841 GGTTCAGCCACTGGAGCTGC 435 2190-2209 coding 104842 GGGCATTGGCCCGGCGGTTC 436 2205-2224 coding 104843 CGCCATTGGCCAGGAGGGCA 437 2220-2239 coding 104844 TATCTCTCAGCTCCACGCCA 438 2235-2254 coding 104845 GCACCACCAGCTGGTTATCT 439 2250-2269 coding 104846 ACAGGCCCTCTGATGGCACC 440 2265-2284 coding 104847 GGGAGTAGATGAGGTACAGG 441 2280-2299 coding 104848 CCTTGAAGAGGACCTGGGAG 442 2295-2314 coding 104849 GAGGAGCACATGGGTGGAGG 443 2327-2346 coding 104850 GCTGATGGTGTGGGTGAGGA 444 2342-2361 coding 104851 GGAGACGGCGATGCGGCTGA 445 2357-2376 coding 104852 GACCTTGGTCTGGTAGGAGA 446 2372-2391 coding 104853 GGCAGAGAGGAGGTTGACCT 447 2387-2406 coding 104854 GCTTGGCCTCAGCCCCCTCT  23 2436-2455 coding 104855 TGGGCTCATACCAGGGCTTG 448 2451-2470 coding 104856 CCCCTCCCAGATAGATGGGC 449 2466-2485 coding 104857 TCTCCAGCTGGAAGACCCCT  92 2481-2500 coding 104858 TGAGTCGGTCACCCTTCTCC 450 2496-2515 coding 104859 GATTGATCTCAGCGCTGAGT 451 2511-2530 coding 104860 CGAGATAGTCGGGCCGATTG 452 2526-2545 coding 104861 CAGACTCGGCAAAGTCGAGA  89 2541-2560 coding 104862 CAAAGTAGACCTGCCCAGAC 453 2556-2575 coding 104863 ACAGGGCAATGATCCCAAAG 454 2571-2590 stop 104864 ATGTTCGTCCTCCTCACAGG 455 2586-2605 stop 104865 GTTTGGGAAGGTTGGATGTT 456 2601-2620 3′-UTR 104866 AAGAGGTTGAGGGTGTCTGA 457 2657-2676 3′-UTR 104867 CTCTTTTTGAGCCAGAAGAG 458 2672-2691 3′-UTR 104868 CCTAAGCCCCCAATTCTCTT 459 2687-2706 3′-UTR 104869 AGCTTGGGTTCCGACCCTAA 460 2702-2721 3′-UTR 104870 TTGCTTAAAGTTCTAAGCTT 461 2717-2736 3′-UTR 104871 GAAGTGGTGGTCTTGTTGCT 462 2732-2751 3′-UTR 104872 TGAATCCCAGGTTTCGAAGT 463 2747-2766 3′-UTR 104873 CAGGCCACACATTCCTGAAT 464 2762-2781 3′-UTR 104874 CAGCACTTCACTGTGCAGGC 465 2777-2796 3′-UTR 104875 ATTCTTAGTGGTTGCCAGCA 466 2792-2811 3′-UTR 104876 GAGGCCCCAGTTTGAATTCT 467 2807-2826 3′-UTR 104877 CCCCAGTGAGTTCTGGAGGC 468 2822-2841 3′-UTR 104878 GATCAAAGCTGTAGGCCCCA 469 2837-2856 3′-UTR 104879 ATTCCAGATGTCAGGGATCA 470 2852-2871 3′-UTR 104880 CTCCCTGGTCTCCAGATTCC 471 2867-2886 3′-UTR 104881 GGCCAGAACCAAAGGCTCCC 472 2882-2901 3′-UTR 104882 GTCCTGCAGCATTCTGGCCA 473 2897-2916 3′-UTR 104883 GTGAGGTCTTCTCAAGTCCT 474 2912-2931 3′-UTR 104884 TGTGTCAATTTCTAGGTGAG 475 2927-2946 3′-UTR 104885 GGCCTAAGGTCCACTTGTGT 476 2942-2961 3′-UTR 104886 ATCTGGAGAGAGGAAGGCCT 477 2957-2976 3′-UTR 104887 AGGAAGTCTGGAAACATCTG 478 2972-2991 3′-UTR 104888 GGGCTCCGTGTCTCAAGGAA 479 2987-3006 3′-UTR 104889 AAATAGAGGGAGCTGGCTCC 480 3019-3038 3′-UTR 104890 CACAAGTGCAAACATAAATA 481 3034-3053 3′-UTR 104891 TCCCAAATAAATACATTCAT 482 3091-3110 3′-UTR 104892 CAGGATACCCCGGTCTCCCA 483 3106-3125 3′-UTR 104893 CTACATTGGGTCCCCCAGGA 484 3121-3140 3′-UTR 104894 GAGCCAAGGCAGCTCCTACA 485 3136-3155 3′-UTR 104895 ACGGAAAACATGTCTGAGCC 486 3151-3170 3′-UTR 104896 TTCAGCTCCGTTTTCACGGA 487 3166-3185 3′-UTR 104897 GGGAACAGCCTATTGTTCAG 488 3181-3200 3′-UTR 104898 TCAAAAGAAGGCACAGAGGC 489 3214-3233 3′-UTR 104899 TTTTAAAAAACATAATCAAA 490 3229-3248 3′-UTR 104900 TTAATCAGATAAATATTTTA 491 3244-3263 3′-UTR 104901 CATTGTTTAGACAACTTAAT 492 3259-3278 3′-UTR 104902 TGGTCACCAAATCAGCATTG 493 3274-3293 3′-UTR 104903 GCAATGAGTGACAGTTGGTC 494 3289-3308 3′-UTR 104904 GGGAGCAGAGGCTCAGCAAT 495 3304-3323 3′-UTR 104905 ATAGTAGGGCGATTACAGAC 496 3336-3355 3′-UTR 104906 ATTTCTCGCCACTGAATAGT 497 3351-3370 3′-UTR

[0328] TABLE 37 Inhibition of Human TNF-α mRNA Expression by Chimeric (deoxy gapped) Phosphorothioate Oligodeoxynucleotides ISIS SEQ ID GENE TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION basal — — 0.0% — induced — — 100.0%  0.0%  28089  69 intron 1 42.3% 57.7% 104649 251 5′-UTR 165.6% — 104650 252 5′-UTR 75.8% 24.2% 104651 253 5′-UTR 58.2% 41.8% 104652 254 5′-UTR 114.5% — 104653 255 5′-UTR 84.9% 15.1% 104654 256 5′-UTR 80.8% 19.2% 104655 257 5′-UTR 94.3%  5.7% 104656 258 5′-UTR 78.4% 21.6% 104657 259 5′-UTR 87.4% 12.6% 104658 260 5′-UTR 213.4% — 104659 261 5′-UTR 96.3%  3.7% 104660 262 5′-UTR 153.1% — 104661 263 5′-UTR 90.0% 10.0% 104662 264 5′-UTR 33.3% 66.7% 104663 265 5′-UTR 144.2% — 104664 266 AUG 76.3% 23.7% 104665 267 AUG 185.3% — 104666 268 AUG 67.4% 32.6% 104667 269 Coding 94.3%  5.7% 104668 270 Coding 63.1% 36.9% 104669 271 Coding 50.8% 49.2% 104670 272 Coding 43.7% 56.3% 104671 273 Coding 52.2% 47.8% 104672 274 Coding 51.8% 48.2% 104673 275 Coding 102.3% — 104674 276 Coding 135.4% — 104675 277 Coding 83.1% 16.9% 104676 278 Coding 87.5% 12.5% 104677 279 Coding 53.6% 46.4% 104678 280 Coding 75.2% 24.8% 104679 281 Coding 114.0% — 104680 282 Coding 142.5% — 104681 283 Coding 58.5% 41.5% 104682 284 Coding 101.9% — 104683 285 Coding 77.1% 22.9% 104684 286 Coding 61.0% 39.0% 104685 287 Coding 65.9% 34.1% 104686 288 E2/I2 59.2% 40.8% 104687 289 Coding 77.0% 23.0% 104688 290 Coding 40.1% 59.9% 104689 291 Coding 78.6% 21.4% 104690 292 Coding 90.9%  9.1% 104691 293 Coding 107.6% — 104692 294 Coding 63.4% 36.6% 104693 295 Coding 74.1% 25.9% 104694 296 Coding 108.3% — 104695 297 Coding 48.2% 51.8% 104696 298 Coding 120.3% — 104697 299 Coding 45.0% 55.0% 104698 300 Coding 77.1% 22.9% 104699 301 Coding 143.7% — 104700 302 Coding 96.1%  3.9% 104701 303 Coding 106.8% — 104702 304 Coding 157.4% — 104703 305 Coding 84.3% 15.7% 104704 306 Coding 182.8% — 104705 307 Coding 125.1% — 104706 308 Coding 81.8% 18.2% 104707 309 Coding 104.8% — 104708 310 Coding 163.0% — 104709 311 Coding 95.0%  5.0% 104710 312 Coding 182.1% — 104711 313 Coding 82.1% 17.9% 104712 314 Coding 118.1% — 104713 315 Coding 31.1% 68.9% 104714 316 Coding 90.5%  9.5% 104715 317 Coding 96.7%  3.3% 104716 318 Coding 180.7% — 104717  93 Coding 71.6% 28.4% 104718  94 Coding 187.0% — 104719 319 Coding 88.8% 11.2% 104720 320 Coding 166.5% — 104721 321 Coding 65.0% 35.0% 104722 322 Coding 59.6% 40.4% 104723 26 Coding 90.1%  9.9% 104724 323 Coding 88.7% 11.3% 104725  90 Coding 94.7%  5.3% 104726  91 Coding 84.1% 15.9% 104727 324 Coding 125.3% — 104728 325 Coding 221.7% — 104729 326 Coding 102.4% — 104730 327 Coding 151.6% — 104731 328 Coding 102.2% — 104732 329 Coding 53.2% 46.8% 104733 330 Stop 57.0% 43.0% 104734  88 Coding 119.2% — 104735 331 3′-UTR 71.2% 28.8% 104736 332 Stop 79.0% 21.0% 104737 333 3′-UTR 87.4% 12.6% 104738 334 Stop 36.8% 63.2% 104739 335 3′-UTR 106.0% — 104740 336 3′-UTR 130.9% — 104741 337 3′-UTR 79.2% 20.8% 104742 338 3′-UTR 159.0% — 104743 339 3′-UTR 96.1%  3.9% 104744 340 3′-UTR 129.9% — 104745 341 3′-UTR 80.2% 19.8% 104746 342 3′-UTR 168.8% — 104747 343 3′-UTR 89.2% 10.8% 104748 344 3′-UTR 103.4% — 104749 345 3′-UTR 89.0% 11.0% 104750 346 3′-UTR 160.0% — 104751 347 3′-UTR 60.1% 39.9% 104752 348 3′-UTR 72.4% 27.6% 104753 349 3′-UTR 70.0% 30.0% 104754 350 3′-UTR 115.6% — 104755 351 3′-UTR 71.7% 28.3% 104756 352 3′-UTR 91.5%  8.5% 104757 353 3′-UTR 85.6% 14.4% 104758 354 3′-UTR 97.6%  2.4% 104759 355 3′-UTR 68.6% 31.4% 104760 356 3′-UTR 182.4% — 104761 357 3′-UTR 110.9% — 104762 358 3′-UTR 161.4% — 104763 359 3′-UTR 102.0% — 104764 360 3′-UTR 113.5% — 104765 361 3′-UTR 154.8% — 104766 362 3′-UTR 126.4% — 104767 363 3′-UTR 116.1% — 104768 364 3′-UTR 177.7% — 104769 365 3′-UTR 89.8% 10.2% 104770 366 3′-UTR 94.3%  5.7% 104771 367 3′-UTR 191.2% — 104772 368 3′-UTR 80.3% 19.7% 104773 369 3′-UTR 133.9% — 104774  34 3′-UTR 94.8%  5.2% 104775 370 3′-UTR 80.6% 19.4% 104776 371 3′-UTR 90.1%  9.9% 104777 372 3′-UTR 84.7% 15.3% 104778 373 3′-UTR 121.3% — 104779 374 3′-UTR 97.8%  2.2% 104780 375 3′-UTR 67.6% 32.4% 104781 376 3′-UTR 141.5% — 104782 377 3′-UTR 96.5%  3.5% 104783 378 3′-UTR 153.2% — 104784 379 3′-UTR 85.4% 14.6% 104785 380 3′-UTR 163.9% — 104786 381 3′-UTR 82.9% 17.1% 104787 382 3′-UTR 89.7% 10.3% 104788 383 3′-UTR 103.9% — 104789 384 3′-UTR 75.8% 24.2% 104790 385 3′-UTR 106.3% — 104791 386 3′-UTR 165.3% — 104792 387 3′-UTR 71.8% 28.2% 104793 388 3′-UTR 101.9% — 104794 389 3′-UTR 70.7% 29.3% 104795 390 3′-UTR 68.8% 31.2% 104796 391 3′-UTR 93.4%  6.6% 104797  37 3′-UTR 131.7% — 104798 392 3′-UTR 89.4% 10.6% 104799 393 3′-UTR 89.6% 10.4% 104800 394 3′-UTR 89.0% 11.0% 104801 395 3′-UTR 196.8% — 104802 396 3′-UTR 189.3% — 104803 397 3′-UTR 119.7% — 104804 398 3′-UTR 102.4% — 104805 399 3′-UTR 90.6%  9.4% 104806 400 3′-UTR 89.1% 10.9% 104807 401 3′-UTR 152.6% — 104808 402 3′-UTR 96.8%  3.2% 104809 403 3′-UTR 178.8% — 104810 404 3′-UTR 94.9%  5.1% 104811 405 3′-UTR 234.4% — 104812 406 3′-UTR 114.3% — 104813 407 3′-UTR 153.7% — 104814 408 3′-UTR 86.3% 13.7% 104815 409 3′-UTR 153.9% — 104816 410 3′-UTR 79.9% 20.1% 104817 411 3′-UTR 196.5% — 104818 412 3′-UTR 94.3%  5.7% 104819 413 3′-UTR 143.3% — 104820 414 3′-UTR 123.8% — 104821 415 3′-UTR 129.2% — 104822 416 5′-UTR 76.6% 23.4% 104823 417 5′-UTR 63.9% 36.1% 104824 418 5′-UTR 22.0% 78.0% 104825 419 5′-UTR 109.4% — 104826 420 5′-UTR 45.2% 54.8% 104827 421 5′-UTR 68.9% 31.1% 104828 422 5′-UTR 70.9% 29.1% 104829 423 AUG 46.6% 53.4% 104830 424 Coding 55.0% 45.0% 104831 425 Coding 49.5% 50.5% 104832 426 Coding 106.0% — 104833 427 Coding 23.7% 76.3% 104834 428 Coding 91.8%  8.2% 104835 429 Coding 72.3% 27.7% 104836 430 Coding 63.4% 36.6% 104837 431 Coding 31.0% 69.0% 104838 432 Coding 18.0% 82.0% 104839 433 Coding 67.9% 32.1% 104840 434 Coding 93.8%  6.2% 104841 435 Coding 43.0% 57.0% 104842 436 Coding 73.2% 26.8% 104843 437 Coding 48.1% 51.9% 104844 438 Coding 39.2% 60.8% 104845 439 Coding 37.6% 62.4% 104846 440 Coding 81.7% 18.3% 104847 441 Coding 50.8% 49.2% 104848 442 Coding 56.7% 43.3% 104849 443 Coding 51.8% 48.2% 104850 444 Coding 91.8%  8.2% 104851 445 Coding 93.9%  6.1% 104852 446 Coding 100.9% — 104853 447 Coding 67.7% 32.3% 104854  23 Coding 11.0% 89.0% 104855 448 Coding 62.5% 37.5% 104856 449 Coding 67.8% 32.2% 104857  92 Coding 28.1% 71.9% 104858 450 Coding 76.2% 23.8% 104859 451 Coding 52.3% 47.7% 104860 452 Coding 93.6% 6.4% 104861  89 Coding 79.3% 20.7% 104862 453 Coding 63.1% 36.9% 104863 454 Stop 64.5% 35.5% 104864 455 Stop 43.2% 56.8% 104865 456 3′-UTR 83.1% 16.9% 104866 457 3′-UTR 49.4% 50.6% 104867 458 3′-UTR 49.5% 50.5% 104868 459 3′-UTR 89.6% 10.4% 104869 460 3′-UTR 21.4% 78.6% 104870 461 3′-UTR 118.0% — 104871 462 3′-UTR 55.8% 44.2% 104872 463 3′-UTR 49.0% 51.0% 104873 464 3′-UTR 92.6%  7.4% 104874 465 3′-UTR 33.4% 66.6% 104875 466 3′-UTR 36.2% 63.8% 104876 467 3′-UTR 73.4% 26.6% 104877 468 3′-UTR 40.9% 59.1% 104878 469 3′-UTR 78.7% 21.3% 104879 470 3′-UTR 75.4% 24.6% 104880 471 3′-UTR 50.2% 49.8% 104881 472 3′-UTR 47.0% 53.0% 104882 473 3′-UTR 82.7% 17.3% 104883 474 3′-UTR 46.4% 53.6% 104884 475 3′-UTR 46.1% 53.9% 104885 476 3′-UTR 156.9% — 104886 477 3′-UTR 102.4% — 104887 478 3′-UTR 59.1% 40.9% 104888 479 3′-UTR 64.7% 35.3% 104889 480 3′-UTR 83.7% 16.3% 104890 481 3′-UTR 52.9% 47.1% 104891 482 3′-UTR 87.9% 12.1% 104892 483 3′-UTR 39.8% 60.2% 104893 484 3′-UTR 71.1% 28.9% 104894 485 3′-UTR 34.0% 66.0% 104895 486 3′-UTR 129.8% — 104896 487 3′-UTR 57.6% 42.4% 104897 488 3′-UTR 49.6% 50.4% 104898 489 3′-UTR 71.7% 28.3% 104899 490 3′-UTR 101.5% — 104900 491 3′-UTR 142.1% — 104901 492 3′-UTR 55.9% 44.1% 104902 493 3′-UTR 85.3% 14.7% 104903 494 3′-UTR 46.0% 54.0% 104904 495 3′-UTR 59.9% 40.1% 104905 496 3′-UTR 47.2% 52.8% 104906 497 3′-UTR 56.3% 43.7%

[0329] Oligonucleotides 104662 (SEQ ID NO: 264), 104669 (SEQ ID NO: 271), 104670 (SEQ ID NO: 272), 104688 (SEQ ID NO: 290), 014695, (SEQ ID NO: 297), 104697 (SEQ ID NO: 299), 104713 (SEQ ID NO: 315), 104738 (SEQ ID NO:334), 104824 (SEQ ID NO: 418), 104826 (SEQ ID NO: 420), 104829 (SEQ ID NO: 423), 104831 (SEQ ID NO: 425), 104833 (SEQ ID NO: 427), 104837 (SEQ ID NO: 431), 104838 (SEQ ID NO: 432), 104841 (SEQ ID NO: 435), 104843 (SEQ ID NO: 437), 104844 (SEQ ID NO: 438), 104845 (SEQ ID NO: 439), 104847 (SEQ ID NO: 441), 104854 (SEQ ID NO: 23), 104857 (SEQ ID NO: 92), 104864 (SEQ ID NO: 455), 104866 (SEQ ID NO: 457), 104867 (SEQ ID NO: 458), 104869 (SEQ ID NO: 460), 104872 (SEQ ID NO: 463), 104874 (SEQ ID NO: 465), 104875 (SEQ ID NO: 466), 104877 (SEQ ID NO: 468), 104880 (SEQ ID NO: 471), 104881 (SEQ ID NO: 472), 104883 (SEQ ID NO: 474), 104884 (SEQ ID NO: 475), 104892 (SEQ ID NO: 483), 104894 (SEQ ID NO: 485), 104897 (SEQ ID NO: 488), 104903 (SEQ ID NO: 494) and 104905 (SEQ ID NO: 496) gave approximately 50% or greater reduction in TNF-α mRNA expression in this assay. Oligonucleotides 104713 (SEQ ID NO: 315), 104824 (SEQ ID NO: 418), 104833 (SEQ ID NO: 427), 104837 (SEQ ID NO: 431), 104838 (SEQ ID NO: 432), 104854 (SEQ ID NO: 23), 104857 (SEQ ID NO: 92), and 104869 (SEQ ID NO: 460) gave approximately 70% or greater reduction in TNFα mRNA expression in this assay.

Example 25 Dose Response of Chimeric (Deoxy Gapped) Antisense Phosphorothioate Oligodeoxynucleotide Effects on TNFα mRNA and Protein Levels

[0330] Several oligonucleotides from the initial screen were chosen for dose response assays. NeoHk cells were grown, treated and processed as described in Example 3. LIPOFECTIN7 was added at a ratio of 3 μg/ml per 100 nM of oligonucleotide. The control included LIPOFECTIN7 at a concentration of 9 μg/ml.

[0331] The human promonocytic leukaemia cell line, THP-1 (American Type Culture Collection, Manassas, Va.) was maintained in RPMI 1640 growth media supplemented with 10% fetal calf serum (FCS; Life Technologies, Rockville, Md.). A total of 8×10⁵ cells were employed for each treatment by combining 50 μl of cell suspension in OPTIMEM™, 1% FBS with oligonucleotide at the indicated concentrations to reach a final volume of 100 μl with OPTIMEM™, 1% FBS. Cells were then transferred to a 1 mm electroporation cuvette and electroporated using an Electrocell Manipulator 600 instrument (Biotechnologies and Experimental Research, Inc.) employing 90 V, 1000 μF, at 13Ω. Electroporated cells were then transferred to 24 well plates. 400 μl of RPMI 1640, 10% FCS was added to the cells and the cells were allowed to recover for 6 hrs. Cells were then induced with LPS at a final concentration of 100 ng/ml for 2 hours. RNA was isolated and processed as described in Example 3. Results with NeoHK cells are shown in Table 38 for mRNA, and Table 39 for protein. Results with THP-1 cells are shown in Table 40.

[0332] Most of the oligonucleotides tested showed dose response effects with a maximum inhibition of mRNA greater than 70% and a maximum inhibition of protein greater than 85%. TABLE 38 Dose Response of NeoHK Cells to TNF-α Chimeric (deoxy gapped) Antisense Oligonucleotides ASO Gene % mRNA % mRNA ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — — 100% —  16798 128 coding  30 nM 87% 13% ″ ″ ″ 100 nM 129% — ″ ″ ″ 300 nM 156% —  21823  69 intron 1  30 nM 82% 18% ″ ″ ″ 100 nM 90% 10% ″ ″ ″ 300 nM 59% 41%  28088  68 intron 1  30 nM 68% 32% ″ ″ ″ 100 nM 43% 57% ″ ″ ″ 300 nM 42% 58%  28089  69 intron 1  30 nM 59% 41% ″ ″ ″ 100 nM 44% 56% ″ ″ ″ 300 nM 38% 62% 104697 299 coding  30 nM 60% 40% ″ ″ ″ 100 nM 45% 55% ″ ″ ″ 300 nM 27% 73% 104777 372 3′-UTR  30 nM 66% 34% ″ ″ ″ 100 nM 55% 45% ″ ″ ″ 300 nM 43% 57%

[0333] TABLE 39 Dose Response of NeoHK Cells to TNF-α Chimeric (deoxy gapped) Antisense Oligonucleotides ASO Gene % Protein % Protein ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — — 100.0% —  16798 128 coding  30 nM 115.0% — ″ ″ ″ 100 nM 136.0% — ″ ″ ″ 300 nM 183.0% —  28089  69 intron 1  30 nM 87.3% 12.7% ″ ″ ″ 100 nM 47.4% 52.6% ″ ″ ″ 300 nM 22.8% 77.2% 104681 283 coding  30 nM 91.3%  8.7% ″ ″ ″ 100 nM 62.0% 38.0% ″ ″ ″ 300 nM 28.5% 71.5% 104697 299 coding  30 nM 87.1% 12.9% ″ ″ ″ 100 nM 59.6% 40.4% ″ ″ ″ 300 nM 29.1% 70.9% 104838 432 coding  30 nM 91.9%  8.1% ″ ″ ″ 100 nM 56.9% 43.1% ″ ″ ″ 300 nM 14.8% 85.2% 104854  23 coding  30 nM 64.4% 35.6% ″ ″ ″ 100 nM 42.3% 57.7% ″ ″ ″ 300 nM 96.1%  3.9% 104869 460 3′-UTR  30 nM 88.9% 11.1% ″ ″ ″ 100 nM 56.8% 43.2% ″ ″ ″ 300 nM 42.3% 57.7%

[0334] TABLE 40 Dose Response of LPS-Induced THP-1 Cells to Chimeric (deoxy gapped) TNF-α Antisense Phosphorothioate Oligodeoxynucleotides (ASOs) ASO Gene % mRNA % mRNA ISIS # SEQ ID NO: Target Dose Expression Inhibition induced — — — 100% —  16798 128 coding  1 μM 102% — ″ ″ ″  3 μM 87% 13% ″ ″ ″ 10 μM 113% — ″ ″ ″ 30 μM 134% —  28089  69 intron 1  1 μM 39% 61% ″ ″ ″  3 μM 79% 21% ″ ″ ″ 10 μM 91%  9% ″ ″ ″ 30 μM 63% 37% 104697 299 coding  1 μM 99%  1% ″ ″ ″  3 μM 96%  4% ″ ″ ″ 10 μM 92%  8% ″ ″ ″ 30 μM 52% 48% 104838 432 coding  1 μM 31% 69% ″ ″ ″  3 μM 20% 80% ″ ″ ″ 10 μM 15% 85% ″ ″ ″ 30 μM 7% 93% 104854  23 coding  1 μM 110% — ″ ″ ″  3 μM 90% 10% ″ ″ ″ 10 μM 95%  5% ″ ″ ″ 30 μM 61% 39%

Example 26 Further Optimization of Human TNF-α Antisense Oligonucleotide Chemistry

[0335] Additional analogs of TNF-α oligonucleotides were designed and synthesized to find an optimum gap size. The sequences and chemistries are shown in Table 36.

[0336] Dose response experiments are performed as described in Example 3. TABLE 41 Nucleotide Sequences of TNF-α Chimeric Backbone (deoxy gapped) Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID CO- TARGET NO. (5′ -> 3′) NO: ORDINATES² REGION 110554 GCTGATTAGAGAGAGGTCCC 432 104838 analog 110555 GCTGATTAGAGAGAGGTCCC ″ 104838 analog 110556 GCTGATTAGAGAGAGGTCCC ″ 104838 analog 110557 GCTGATTAGAGAGAGGTCCC ″ 104838 analog 110583 GCTGATTAGAGAGAGGTCCC ″ 104838 analog 110558 CTGATTAGAGAGAGGTCCC 498 1596-1614 coding 110559 CTGATTAGAGAGAGGTCCC ″ ″ ″ 110560 CTGATTAGAGAGAGGTCCC ″ ″ ″ 110561 CTGATTAGAGAGAGGTCCC ″ ″ ″ 110562 CTGATTAGAGAGAGGTCCC ″ ″ ″ 110563 CTGATTAGAGAGAGGTCCC ″ ″ ″ 110564 CTGATTAGAGAGAGGTCCC ″ ″ ″ 110565 CTGATTAGAGAGAGGTCCC ″ ″ ″ 110566 CTGATTAGAGAGAGGTCCC ″ ″ ″ 110567 CTGATTAGAGAGAGGTCCC ″ ″ ″ 110584 CTGATTAGAGAGAGGTCCC ″ ″ ″ 108371 CTGATTAGAGAGAGGTCC 499 1597-1614 coding 110568 CTGATTAGAGAGAGGTCC ″ ″ ″ 110569 CTGATTAGAGAGAGGTCC ″ ″ ″ 110570 CTGATTAGAGAGAGGTCC ″ ″ ″ 110585 CTGATTAGAGAGAGGTCC ″ ″ ″ 110571 CTGGTTATCTCTCAGCTCCA 299 104697 analog 110572 CTGGTTATCTCTCAGCTCCA ″ 104697 analog 110573 CTGGTTATCTCTCAGCTCCA ″ 104697 analog 110586 CTGGTTATCTCTCAGCTCCA ″ 104697 analog 110574 GATCACTCCAAAGTGCAGCA 283 104681 analog 110575 GATCACTCCAAAGTGCAGCA ″ 104681 analog 110576 GATCACTCCAAAGTGCAGCA ″ 104681 analog 110587 GATCACTCCAAAGTGCAGCA ″ 104681 analog 110577 AGCTTGGGTTCCGACCCTAA 460 104689 analog 110578 AGCTTGGGTTCCGACCCTAA ″ 104689 analog 110579 AGCTTGGGTTCCGACCCTAA ″ 104689 analog 110588 AGCTTGGGTTCCGACCCTAA ″ 104689 analog 110580 AGGTTGACCTTGGTCTGGTA 315 104713 analog 110581 AGGTTGACCTTGGTCTGGTA ″ 104713 analog 110582 AGGTTGACCTTGGTCTGGTA ″ 104713 analog 110589 AGGTTGACCTTGGTCTGGTA ″ 104713 analog 110637 GTGTGCCAGACACCCTATCT  69 21823 analog 110651 GTGTGCCAGACACCCTATCT ″ 21823 analog 110665 GTGTGCCAGACACCCTATCT ″ 21823 analog 110679 GTGTGCCAGACACCCTATCT ″ 21823 analog 110693 GTGTGCCAGACACCCTATCT ″ 21823 analog 110707 GTGTGCCAGACACCCTATCT ″ 21823 analog 110590 TGAGTGTCTTCTGTGTGCCA 500 1411-1430 intron 1 110597 TGAGTGTCTTCTGTGTGCCA ″ ″ ″ 110604 TGAGTGTCTTCTGTGTGCCA ″ ″ ″ 110611 TGAGTGTCTTCTGTGTGCCA ″ ″ ″ 110618 TGAGTGTCTTCTGTGTGCCA ″ ″ ″ 110625 TGAGTGTCTTCTGTGTGCCA ″ ″ ″ 110591 GAGTGTCTTCTGTGTGCCAG 501 1410-1429 intron 1 110598 GAGTGTCTTCTGTGTGCCAG ″ ″ ″ 110605 GAGTGTCTTCTGTGTGCCAG ″ ″ ″ 110612 GAGTGTCTTCTGTGTGCCAG ″ ″ ″ 110619 GAGTGTCTTCTGTGTGCCAG ″ ″ ″ 110626 GAGTGTCTTCTGTGTGCCAG ″ ″ ″ 110592 AGTGTCTTCTGTGTGCCAGA 144 100181 analog 110599 AGTGTCTTCTGTGTGCCAGA ″ 100181 analog 110606 AGTGTCTTCTGTGTGCCAGA ″ 100181 analog 110613 AGTGTCTTCTGTGTGCCAGA ″ 100181 analog 110620 AGTGTCTTCTGTGTGCCAGA ″ 100181 analog 110627 AGTGTCTTCTGTGTGCCAGA ″ 100181 analog 110593 GTGTCTTCTGTGTGCCAGAC 145 100182 analog 110600 GTGTCTTCTGTGTGCCAGAC ″ 100182 analog 110607 GTGTCTTCTGTGTGCCAGAC ″ 100182 analog 110614 GTGTCTTCTGTGTGCCAGAC ″ 100182 analog 110621 GTGTCTTCTGTGTGCCAGAC ″ 100182 analog 110628 GTGTCTTCTGTGTGCCAGAC ″ 100182 analog 110594 TGTCTTCTGTGTGCCAGACA 146 100183 analog 110601 TGTCTTCTGTGTGCCAGACA ″ 100183 analog 110608 TGTCTTCTGTGTGCCAGACA ″ 100183 analog 110615 TGTCTTCTGTGTGCCAGACA ″ 100183 analog 110622 TGTCTTCTGTGTGCCAGACA ″ 100183 analog 110629 TGTCTTCTGTGTGCCAGACA ″ 100183 analog 110595 GTCTTCTGTGTGCCAGACAC 147 100184 analog 110602 GTCTTCTGTGTGCCAGACAC ″ 100184 analog 110609 GTCTTCTGTGTGCCAGACAC ″ 100184 analog 110616 GTCTTCTGTGTGCCAGACAC ″ 100184 analog 110623 GTCTTCTGTGTGCCAGACAC ″ 100184 analog 110630 GTCTTCTGTGTGCCAGACAC ″ 100184 analog 110596 TCTTCTGTGTGCCAGACACC 148 100185 analog 110603 TCTTCTGTGTGCCAGACACC ″ 100185 analog 110610 TCTTCTGTGTGCCAGACACC ″ 100185 analog 110617 TCTTCTGTGTGCCAGACACC ″ 100185 analog 110624 TCTTCTGTGTGCCAGACACC ″ 100185 analog 110631 TCTTCTGTGTGCCAGACACC ″ 100185 analog 110632 CTTCTGTGTGCCAGACACCC 149 100186 analog 110646 CTTCTGTGTGCCAGACACCC ″ 100186 analog 110660 CTTCTGTGTGCCAGACACCC ″ 100186 analog 110674 CTTCTGTGTGCCAGACACCC ″ 100186 analog 110688 CTTCTGTGTGCCAGACACCC ″ 100186 analog 110702 CTTCTGTGTGCCAGACACCC ″ 100186 analog 110633 TTCTGTGTGCCAGACACCCT 150 100187 analog 110647 TTCTGTGTGCCAGACACCCT ″ 100187 analog 110661 TTCTGTGTGCCAGACACCCT ″ 100187 analog 110675 TTCTGTGTGCCAGACACCCT ″ 100187 analog 110689 TTCTGTGTGCCAGACACCCT ″ 100187 analog 110703 TTCTGTGTGCCAGACACCCT ″ 100187 analog 110634 TCTGTGTGCCAGACACCCTA 151 100188 analog 110648 TCTGTGTGCCAGACACCCTA ″ 100188 analog 110662 TCTGTGTGCCAGACACCCTA ″ 100188 analog 110676 TCTGTGTGCCAGACACCCTA ″ 100188 analog 110690 TCTGTGTGCCAGACACCCTA ″ 100188 analog 110704 TCTGTGTGCCAGACACCCTA ″ 100188 analog 110635 CTGTGTGCCAGACACCCTAT 152 100189 analog 110649 CTGTGTGCCAGACACCCTAT ″ 100189 analog 110663 CTGTGTGCCAGACACCCTAT ″ 100189 analog 110677 CTGTGTGCCAGACACCCTAT ″ 100189 analog 110691 CTGTGTGCCAGACACCCTAT ″ 100189 analog 110705 CTGTGTGCCAGACACCCTAT ″ 100189 analog 110636 TGTGTGCCAGACACCCTATC 153 100190 analog 110650 TGTGTGCCAGACACCCTATC ″ 100190 analog 110664 TGTGTGCCAGACACCCTATC ″ 100190 analog 110678 TGTGTGCCAGACACCCTATC ″ 100190 analog 110692 TGTGTGCCAGACACCCTATC ″ 100190 analog 110706 TGTGTGCCAGACACCCTATC ″ 100190 analog 110638 TGTGCCAGACACCCTATCTT 154 100191 analog 110652 TGTGCCAGACACCCTATCTT ″ 100191 analog 110666 TGTGCCAGACACCCTATCTT ″ 100191 analog 110680 TGTGCCAGACACCCTATCTT ″ 100191 analog 110694 TGTGCCAGACACCCTATCTT ″ 100191 analog 110708 TGTGCCAGACACCCTATCTT ″ 100191 analog 110639 GTGCCAGACACCCTATCTTC 155 100192 analog 110653 GTGCCAGACACCCTATCTTC ″ 100192 analog 110667 GTGCCAGACACCCTATCTTC ″ 100192 analog 110681 GTGCCAGACACCCTATCTTC ″ 100192 analog 110695 GTGCCAGACACCCTATCTTC ″ 100192 analog 110709 GTGCCAGACACCCTATCTTC ″ 100192 analog 110640 TGCCAGACACCCTATCTTCT 156 100193 analog 110654 TGCCAGACACCCTATCTTCT ″ 100193 analog 110668 TGCCAGACACCCTATCTTCT ″ 100193 analog 110682 TGCCAGACACCCTATCTTCT ″ 100193 analog 110696 TGCCAGACACCCTATCTTCT ″ 100193 analog 110710 TGCCAGACACCCTATCTTCT ″ 100193 analog 110641 GCCAGACACCCTATCTTCTT 157 100194 analog 110655 GCCAGACACCCTATCTTCTT ″ 100194 analog 110669 GCCAGACACCCTATCTTCTT ″ 100194 analog 110683 GCCAGACACCCTATCTTCTT ″ 100194 analog 110697 GCCAGACACCCTATCTTCTT ″ 100194 analog 110711 GCCAGACACCCTATCTTCTT ″ 100194 analog 110642 CCAGACACCCTATCTTCTTC 158 100195 analog 110656 CCAGACACCCTATCTTCTTC ″ 100195 analog 110670 CCAGACACCCTATCTTCTTC ″ 100195 analog 110684 CCAGACACCCTATCTTCTTC ″ 100195 analog 110698 CCAGACACCCTATCTTCTTC ″ 100195 analog 110712 CCAGACACCCTATCTTCTTC ″ 100195 analog 110643 CAGACACCCTATCTTCTTCT 159 100196 analog 110657 CAGACACCCTATCTTCTTCT ″ 100196 analog 110671 CAGACACCCTATCTTCTTCT ″ 100196 analog 110685 CAGACACCCTATCTTCTTCT ″ 100196 analog 110699 CAGACACCCTATCTTCTTCT ″ 100196 analog 110713 CAGACACCCTATCTTCTTCT 100196 analog 110644 AGACACCCTATCTTCTTCTC 160 100197 analog 110658 AGACACCCTATCTTCTTCTC ″ 100197 analog 110672 AGACACCCTATCTTCTTCTC ″ 100197 analog 110686 AGACACCCTATCTTCTTCTC ″ 100197 analog 110700 AGACACCCTATCTTCTTCTC ″ 100197 analog 110714 AGACACCCTATCTTCTTCTC ″ 100197 analog 110645 GACACCCTATCTTCTTCTCT 161 100198 analog 110659 GACACCGTATCTTCTTCTCT ″ 100198 analog 110673 GACACCCTATCTTCTTCTCT ″ 100198 analog 110687 GACACCCTATCTTCTTCTCT ″ 100198 analog 110701 GACACCCTATCTTCTTCTCT ″ 100198 analog 110715 GACACCCTATCTTCTTCTCT ″ 100198 analog

Example 26 Effect of TNFα Antisense Oligonucleotides in TNF-α Transgenic Mouse Models

[0337] The effect of TNF-α antisense oligonucleotides is studied in transgenic mouse models of human diseases. Such experiments can be performed through contract laboratories (e.g., The Laboratory of Molecular Genetics at The Hellenic Pasteur Institute, Athens, Greece) where such transgenic mouse models are available. Such models are available for testing human oligonucleotides in arthritis (Keffer, J., et al., EMBO J., 1991, 10, 4025-4031) and multiple sclerosis (Akassoglou et al., J. Immunol., 1997, 158, 438-445) models. A model for inflammatory bowel disease is available for testing mouse oligonucleotides (Kontoyiannis et al., Immunity, 1999, 10, 387-398).

[0338] Briefly, litters of the appropriate transgenic mouse strain are collected and weighed individually. Twice weekly from birth, oligonucleotide in saline is administered intraperitoneally or intravenously. Injections continue for 7 weeks. Each week the animals are scored for manifestations of the appropriate disease. After the final treatment, the mice are sacrificed and histopathology is performed for indicators of disease as indicated in the references cited for each model.

Example 27 Design and Screening of Duplexed Antisense Compounds Targeting TNFα

[0339] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target TNF-α. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an oligonucleotide to TNFα as described herein. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini. For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:   cgagaggcggacgggaccgTT Antisense Strand   ||||||||||||||||||| TTgctctccgcctgccctggc Complement

[0340] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15 uL of a 5× solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.

[0341] Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate TNF-α expression according to the protocols described herein.

Example 28 Design of Phenotypic Assays and in vivo Studies for the Use of TNF-α Inhibitors

[0342] Phenotypic Assays

[0343] Once TNFα inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition.

[0344] Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of TNF-α in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

[0345] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with TNF-α inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

[0346] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest. Analysis of the geneotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the TNF-α inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 503 <210> SEQ ID NO 1 <211> LENGTH: 3634 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (796..981,1589..1634,1822..1869,2171..2592) <220> FEATURE: <221> NAME/KEY: exon <222> LOCATION: (615)..(981) <220> FEATURE: <221> NAME/KEY: intron <222> LOCATION: (982)..(1588) <220> FEATURE: <221> NAME/KEY: exon <222> LOCATION: (1589)..(1634) <220> FEATURE: <221> NAME/KEY: intron <222> LOCATION: (1635)..(1821) <220> FEATURE: <221> NAME/KEY: exon <222> LOCATION: (1822)..(1869) <220> FEATURE: <221> NAME/KEY: intron <222> LOCATION: (1870)..(2070) <220> FEATURE: <221> NAME/KEY: exon <222> LOCATION: (2171)..(3381) <300> PUBLICATION INFORMATION: <301> AUTHORS: Nedwin, G.E. Naylor, S.L. Sakaguchi, A.Y. Smith, D. Jarrett-Nedwin, J. Pennica, D. Goeddel, D.V. Gray, P.W. <302> TITLE: Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization <303> JOURNAL: Nucleic Acids Res. <304> VOLUME: 13 <305> ISSUE: 17 <306> PAGES: 6361-6373 <307> DATE: 1985-09-11 <308> DATABASE ACCESSION NUMBER: X02910 Genbank <309> DATABASE ENTRY DATE: 1997-02-17 <400> SEQUENCE: 1 gaattccggg tgatttcact cccggctgtc caggcttgtc ctgctacccc acccagcctt 60 tcctgaggcc tcaagcctgc caccaagccc ccagctcctt ctccccgcag gacccaaaca 120 caggcctcag gactcaacac agcttttccc tccaacccgt tttctctccc tcaacggact 180 cagctttctg aagcccctcc cagttctagt tctatctttt tcctgcatcc tgtctggaag 240 ttagaaggaa acagaccaca gacctggtcc ccaaaagaaa tggaggcaat aggttttgag 300 gggcatgggg acggggttca gcctccaggg tcctacacac aaatcagtca gtggcccaga 360 agacccccct cggaatcgga gcagggagga tggggagtgt gaggggtatc cttgatgctt 420 gtgtgtcccc aactttccaa atccccgccc ccgcgatgga gaagaaaccg agacagaagg 480 tgcagggccc actaccgctt cctccagatg agctcatggg tttctccacc aaggaagttt 540 tccgctggtt gaatgattct ttccccgccc tcctctcgcc ccagggacat ataaaggcag 600 ttgttggcac acccagccag cagacgctcc ctcagcaagg acagcagagg accagctaag 660 agggagagaa gcaactacag accccccctg aaaacaaccc tcagacgcca catcccctga 720 caagctgcca ggcaggttct cttcctctca catactgacc cacggcttca ccctctctcc 780 cctggaaagg acacc atg agc act gaa agc atg atc cgg gac gtg gag ctg 831 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu 1 5 10 gcc gag gag gcg ctc ccc aag aag aca ggg ggg ccc cag ggc tcc agg 879 Ala Glu Glu Ala Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg 15 20 25 cgg tgc ttg ttc ctc agc ctc ttc tcc ttc ctg atc gtg gca ggc gcc 927 Arg Cys Leu Phe Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala 30 35 40 acc acg ctc ttc tgc ctg ctg cac ttt gga gtg atc ggc ccc cag agg 975 Thr Thr Leu Phe Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg 45 50 55 60 gaa gag gtgagtgcct ggccagcctt catccactct cccacccaag gggaaatgag 1031 Glu Glu agacgcaaga gagggagaga gatgggatgg gtgaaagatg tgcgctgata gggagggatg 1091 agagagaaaa aaacatggag aaagacgggg atgcagaaag agatgtggca agagatgggg 1151 aagagagaga gagaaagatg gagagacagg atgtctggca catggaaggt gctcactaag 1211 tgtgtatgga gtgaatgaat gaatgaatga atgaacaagc agatatataa ataagatatg 1271 gagacagatg tggggtgtga gaagagagat gggggaagaa acaagtgata tgaataaaga 1331 tggtgagaca gaaagagcgg gaaatatgac agctaaggag agagatgggg gagataagga 1391 gagaagaaga tagggtgtct ggcacacaga agacactcag ggaaagagct gttgaatgct 1451 ggaaggtgaa tacacagatg aatggagaga gaaaaccaga cacctcaggg ctaagagcgc 1511 aggccagaca ggcagccagc tgttcctcct ttaagggtga ctccctcgat gttaaccatt 1571 ctccttctcc ccaacag ttc ccc agg gac ctc tct cta atc agc cct ctg 1621 Phe Pro Arg Asp Leu Ser Leu Ile Ser Pro Leu 65 70 gcc cag gca gtc agtaagtgtc tccaaacctc tttcctaatt ctgggtttgg 1673 Ala Gln Ala Val 75 gtttgggggt agggttagta ccggtatgga agcagtgggg gaaatttaaa gttttggtct 1733 tgggggagga tggatggagg tgaaagtagg ggggtatttt ctaggaagtt taagggtctc 1793 agctttttct tttctctctc ctcttca gga tca tct tct cga acc ccg agt gac 1847 Arg Ser Ser Ser Arg Thr Pro Ser Asp 80 85 aag cct gta gcc cat gtt gta ggtaagagct ctgaggatgt gtcttggaac 1898 Lys Pro Val Ala His Val Val 90 ttggagggct aggatttggg gattgaagcc cggctgatgg taggcagaac ttggagacaa 1958 tgtgagaagg actcgctgag ctcaagggaa gggtggagga acagcacagg ccttagtggg 2018 atactcagaa cgtcatggcc aggtgggatg tgggatgaca gacagagagg acaggaaccg 2078 gatgtggggt gggcagagct cgagggccag gatgtggaga gtgaaccgac atggccacac 2138 tgactctcct ctccctctct ccctccctcc a gca aac cct caa gct gag ggg 2190 Ala Asn Pro Gln Ala Glu Gly 95 100 cag ctc cag tgg ctg aac cgc cgg gcc aat gcc ctc ctg gcc aat ggc 2238 Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly 105 110 115 gtg gag ctg aga gat aac cag ctg gtg gtg cca tca gag ggc ctg tac 2286 Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr 120 125 130 ctc atc tac tcc cag gtc ctc ttc aag ggc caa ggc tgc ccc tcc acc 2334 Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr 135 140 145 cat gtg ctc ctc acc cac acc atc agc cgc atc gcc gtc tcc tac cag 2382 His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln 150 155 160 acc aag gtc aac ctc ctc tct gcc atc aag agc ccc tgc cag agg gag 2430 Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu 165 170 175 180 acc cca gag ggg gct gag gcc aag ccc tgg tat gag ccc atc tat ctg 2478 Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu 185 190 195 gga ggg gtc ttc cag ctg gag aag ggt gac cga ctc agc gct gag atc 2526 Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile 200 205 210 aat cgg ccc gac tat ctc gac ttt gcc gag tct ggg cag gtc tac ttt 2574 Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln Val Tyr Phe 215 220 225 ggg atc att gcc ctg tga ggaggacgaa catccaacct tcccaaacgc 2622 Gly Ile Ile Ala Leu 230 ctcccctgcc ccaatccctt tattaccccc tccttcagac accctcaacc tcttctggct 2682 caaaaagaga attgggggct tagggtcgga acccaagctt agaactttaa gcaacaagac 2742 caccacttcg aaacctggga ttcaggaatg tgtggcctgc acagtgaagt gctggcaacc 2802 actaagaatt caaactgggg cctccagaac tcactggggc ctacagcttt gatccctgac 2862 atctggaatc tggagaccag ggagcctttg gttctggcca gaatgctgca ggacttgaga 2922 agacctcacc tagaaattga cacaagtgga ccttaggcct tcctctctcc agatgtttcc 2982 agacttcctt gagacacgga gcccagccct ccccatggag ccagctccct ctatttatgt 3042 ttgcacttgt gattatttat tatttattta ttatttattt atttacagat gaatgtattt 3102 atttgggaga ccggggtatc ctgggggacc caatgtagga gctgccttgg ctcagacatg 3162 ttttccgtga aaacggagct gaacaatagg ctgttcccat gtagccccct ggcctctgtg 3222 ccttcttttg attatgtttt ttaaaatatt tatctgatta agttgtctaa acaatgctga 3282 tttggtgacc aactgtcact cattgctgag cctctgctcc ccaggggagt tgtgtctgta 3342 atcgccctac tattcagtgg cgagaaataa agtttgctta gaaaagaaac atggtctcct 3402 tcttggaatt aattctgcat ctgcctcttc ttgtgggtgg gaagaagctc cctaagtcct 3462 ctctccacag gctttaagat ccctcggacc cagtcccatc cttagactcc tagggccctg 3522 gagaccctac ataaacaaag cccaacagaa tattccccat cccccaggaa acaagagcct 3582 gaacctaatt acctctccct cagggcatgg gaatttccaa ctctgggaat tc 3634 <210> SEQ ID NO 2 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 2 catgctttca gtgctcat 18 <210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 3 tgagggagcg tctgctggct 20 <210> SEQ ID NO 4 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 4 gtgctcatgg tgtcctttcc 20 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 5 taatcacaag tgcaaacata 20 <210> SEQ ID NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 6 taccccggtc tcccaaataa 20 <210> SEQ ID NO 7 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 7 agcaccgcct ggagccct 18 <210> SEQ ID NO 8 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 8 gctgaggaac aagcaccgcc 20 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 9 aggcagaaga gcgtggtggc 20 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 10 aaagtgcagc aggcagaaga 20 <210> SEQ ID NO 11 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 11 ttagagagag gtccctgg 18 <210> SEQ ID NO 12 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 12 tgactgcctg ggccagag 18 <210> SEQ ID NO 13 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 13 gggttcgaga agatgatc 18 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 14 gggctacagg cttgtcactc 20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 15 cccctcagct tgagggtttg 20 <210> SEQ ID NO 16 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 16 ccattggcca ggagggcatt 20 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 17 accaccagct ggttatctct 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 18 ctgggagtag atgaggtaca 20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 19 cccttgaaga ggacctggga 20 <210> SEQ ID NO 20 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 20 ggtgtgggtg aggagcacat 20 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 21 gtctggtagg agacggcgat 20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 22 gcagagagga ggttgacctt 20 <210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 23 gcttggcctc agccccctct 20 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 24 cctcccagat agatgggctc 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 25 cccttctcca gctggaagac 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 26 atctcagcgc tgagtcggtc 20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 27 tcgagatagt cgggccgatt 20 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 28 aagtagacct gcccagactc 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 29 ggatgttcgt cctcctcaca 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 30 accctaagcc cccaattctc 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 31 ccacacattc ctgaatccca 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 32 aggccccagt gagttctgga 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 33 gtctccagat tccagatgtc 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 34 ctcaagtcct gcagcattct 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 35 tgggtccccc aggatacccc 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 36 acggaaaaca tgtctgagcc 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 37 ctccgttttc acggaaaaca 20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 38 gcctattgtt cagctccgtt 20 <210> SEQ ID NO 39 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 39 ggtcaccaaa tcagcattgt t 21 <210> SEQ ID NO 40 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 40 gaggctcagc aatgagtgac 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: control sequence <400> SEQUENCE: 41 gcccaagctg gcatccgtca 20 <210> SEQ ID NO 42 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: control seqeuence <400> SEQUENCE: 42 gccgaggtcc atgtcgtacg c 21 <210> SEQ ID NO 43 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 43 caggcggtgc ttgttcct 18 <210> SEQ ID NO 44 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 44 gccagagggc tgattagaga ga 22 <210> SEQ ID NO 45 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR probe <400> SEQUENCE: 45 cttctccttc ctgatcgtgg caggc 25 <210> SEQ ID NO 46 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 46 gaaggtgaag gtcggagtc 19 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 47 gaagatggtg atgggatttc 20 <210> SEQ ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR probe <400> SEQUENCE: 48 caagcttccc gttctcagcc 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: control sequence <400> SEQUENCE: 49 tctgagtagc agaggagctc 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 50 tgcgtctctc atttcccctt 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 51 tcccatctct ctccctctct 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 52 cagcgcacat ctttcaccca 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 53 tctctctcat ccctccctat 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 54 cgtctttctc catgtttttt 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 55 cacatctctt tctgcatccc 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 56 ctctcttccc catctcttgc 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 57 gtctctccat ctttccttct 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 58 ttccatgtgc cagacatcct 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 59 atacacactt agtgagcacc 20 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 60 ttcattcatt cattcactcc 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 61 tatatctgct tgttcattca 20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 62 ctgtctccat atcttattta 20 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 63 tctcttctca caccccacat 20 <210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 64 cacttgtttc ttcccccatc 20 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 65 ctcaccatct ttattcatat 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 66 atatttcccg ctctttctgt 20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 67 catctctctc cttagctgtc 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 68 tcttctctcc ttatctcccc 20 <210> SEQ ID NO 69 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 69 gtgtgccaga caccctatct 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 70 tctttccctg agtgtcttct 20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 71 accttccagc attcaacagc 20 <210> SEQ ID NO 72 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 72 ctccattcat ctgtgtattc 20 <210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 73 tgaggtgtct ggttttctct 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 74 acacatcctc agagctctta 20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 75 ctagccctcc aagttccaag 20 <210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 76 cgggcttcaa tccccaaatc 20 <210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 77 aagttctgcc taccatcagc 20 <210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 78 gtccttctca cattgtctcc 20 <210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 79 ccttcccttg agctcagcga 20 <210> SEQ ID NO 80 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 80 ggcctgtgct gttcctccac 20 <210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 81 cgttctgagt atcccactaa 20 <210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 82 cacatcccac ctggccatga 20 <210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 83 gtcctctctg tctgtcatcc 20 <210> SEQ ID NO 84 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 84 ccaccccaca tccggttcct 20 <210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 85 tcctggccct cgagctctgc 20 <210> SEQ ID NO 86 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 86 atgtcggttc actctccaca 20 <210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 87 agaggagagt cagtgtggcc 20 <210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 88 gatcccaaag tagacctgcc 20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 89 cagactcggc aaagtcgaga 20 <210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 90 tagtcgggcc gattgatctc 20 <210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 91 agcgctgagt cggtcaccct 20 <210> SEQ ID NO 92 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 92 tctccagctg gaagacccct 20 <210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 93 cccagataga tgggctcata 20 <210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 94 ccagggcttg gcctcagccc 20 <210> SEQ ID NO 95 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 95 cctctggggt ctccctctgg 20 <210> SEQ ID NO 96 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 96 caggggctct tgatggcaga 20 <210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 97 gaggaggttg accttggtct 20 <210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 98 ggtaggagac ggcgatgcgg 20 <210> SEQ ID NO 99 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 99 ctgatggtgt gggtgaggag 20 <210> SEQ ID NO 100 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 100 aggcactcac ctcttccctc 20 <210> SEQ ID NO 101 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 101 ccctggggaa ctgttgggga 20 <210> SEQ ID NO 102 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 102 agacacttac tgactgcctg 20 <210> SEQ ID NO 103 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 103 gaagatgatc ctgaagagga 20 <210> SEQ ID NO 104 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 104 gagctcttac ctacaacatg 20 <210> SEQ ID NO 105 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 105 tgagggtttg ctggagggag 20 <210> SEQ ID NO 106 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: control sequence <400> SEQUENCE: 106 gatcgcgtcg gactatgaag 20 <210> SEQ ID NO 107 <211> LENGTH: 7208 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (4527..4712,5225..5279,5457..5504,5799..6217) <220> FEATURE: <221> NAME/KEY: exon <222> LOCATION: (4371)..(4712) <220> FEATURE: <221> NAME/KEY: intron <222> LOCATION: (4713)..(5224) <220> FEATURE: <221> NAME/KEY: exon <222> LOCATION: (5225)..(5279) <220> FEATURE: <221> NAME/KEY: intron <222> LOCATION: (5280)..(5456) <220> FEATURE: <221> NAME/KEY: exon <222> LOCATION: (5457)..(5504) <220> FEATURE: <221> NAME/KEY: intron <222> LOCATION: (5505)..(5798) <220> FEATURE: <221> NAME/KEY: exon <222> LOCATION: (5799)..(>6972) <300> PUBLICATION INFORMATION: <301> AUTHORS: Semon, D. Kawashima, E. Jongeneel, C.V. Shakhov, A.N. Nedospasov, S.A. <302> TITLE: Nucleotide sequence of the murine TNF locus, including the TNF-alpha (tumor necrosis factor) and TNF-beta (lymphotoxin) genes <303> JOURNAL: Nucleic Acids Res. <304> VOLUME: 15 <305> ISSUE: 21 <306> PAGES: 9083-9084 <307> DATE: 1987-11-11 <308> DATABASE ACCESSION NUMBER: Y00467 Genbank <309> DATABASE ENTRY DATE: 1993-05-11 <400> SEQUENCE: 107 gaattctgaa gctccctctg tacagagcat tggaagcctg gggtgtacat ttggggttac 60 atgatcttgg ggttctaaga gaataccccc aaatcatctt ccagacctgg aacattctag 120 gacagggttc tcaaccttcc taactccatg accctttaat acagttcctc atgttgtggt 180 gaccccaacc atacaattat tttcgttgct atttcataac tgtaatttcg ctgctattat 240 gaatcataat gtaaatattt gttttaaata gaggtttgcc aaagggacct tgcccacagg 300 ttgagaactg ccgctccaga gagtaagggg acacagttaa gattgttaca caccaggatg 360 ccccagattt ggggagaggg cactgtaatg gaacttcttg acatgaaact ggcagatgaa 420 actggcagaa aaaaaaaaaa aagctgggca gtggtggcac acacctttaa tcccagcact 480 tgggaggcag aggcaggcgg atttctgagt tctaggccag cctggtctac agagtgagtt 540 tcaggacagc cagggctaca cagagaaacc ctgtctcgaa aaaagcaaaa aaaaaaaaaa 600 aaaaaaaaaa aaactggcag atgaccagaa aatacagata tattggaata actgtgactt 660 gaacccccaa agacaagaga ggaaataggc ctgaaggggc ggcaggcatg tcaagcatcc 720 agagccctgg gttcgaacct gaaaaaacaa aggtgccgct aaccacatgt ggcttcggag 780 ccctccagac atgaccatga tcgacagaga gggaaatgtg cagagaagcc tgtgagcagt 840 caagggtgca gaagtgatat aaaccatcac tcttcaggga accaggcttc cagtcacagc 900 ccagctgcac cctctccacg aattgctcgg ccgttcactg gaactcctgg gcctgaccca 960 gctccctgct agtccctgcg gcccacagtt ccccggaccc gactcccttt cccagaacgc 1020 agtagtctaa gcccttagcc tgcggttctc tcctaggccc cagcctttcc tgccttcgac 1080 tgaaacagca gcatcttcta agccctgggg gcttccccaa gccccagccc cgacctagaa 1140 cccgcccgct gcctgccaca ctgccgcttc ctctataaag ggacccgagc gccagcgccc 1200 aggaccccgc acagcaggtg agcctctcct accctgtctc cttgggctta ccctggtatc 1260 aggcatccct caggatccta cctcctttct tgagccacag ccttttctat acaacctgcc 1320 tggatcccca gccttaatgg gtctggtcct cctgtcgtgg ctttgatttt tggtctgttc 1380 ctgtggcggc cttatcagtc tctctctctc tctctctctc tctctctctc tctctctctc 1440 tctctctctc tctccctctc tctctctctc tctctctctc ttctctctct ctgcctctgt 1500 tagccattgt ctgattctat ggtggagctt tcctcttccc ctctgtctct ccttatccct 1560 gctcacttca gggttcccct gcctgtcccc ttttctgtct gtcgccctgt ctctcagggt 1620 ggctgtctca gctgggaggt aaggtctgtc ttccgctgtg tgccccgcct ccgctacaca 1680 cacacactct ctctctctct ctcagcaggt tctccacatg acactgctcg gccgtctcca 1740 cctcttgagg gtgcttggca cccctcctgt cttcctcctg gggctgctgc tggccctgcc 1800 tctaggggcc caggtgaggc agcaagagat tgggggtgct ggggtggcct agctaactca 1860 gagtcctaga gtcctctcca ctctcttctg tcccagggac tctctggtgt ccgcttctcc 1920 gctgccagga cagcccatcc actccctcag aagcacttga cccatggcat cctgaaacct 1980 gctgctcacc ttgttggtaa acttctgcct ccagaggaga ggtccagtcc ctgccttttg 2040 tcctacttgc ccaggggctc aggcgatctt cccatctccc cacaccaact tttcttaccc 2100 ctaagggcag gcaccccact cccatctccc taccaaccat cccacttgtc cagtgcctgc 2160 tcctcaggga tggggacctc tgatcttgat agccccccaa tgtcttgtgc ctcttcccag 2220 ggtaccccag caagcagaac tcactgctct ggagagcaag cacggatcgt gcctttctcc 2280 gacatggctt ctctttgagc aacaactccc tcctgatccc caccagtggc ctctactttg 2340 tctactccca ggtggttttc tctggagaaa gctgctcccc cagggccatt cccactccca 2400 tctacctggc acacgaggtc cagctctttt cctcccaata ccccttccat gtgcctctcc 2460 tcagtgcgca gaagtctgtg tatccgggac ttcaaggacc gtgggtgcgc tcaatgtacc 2520 agggggctgt gttcctgctc agtaagggag accagctgtc cacccacacc gacggcatct 2580 cccatctaca cttcagcccc agcagtgtat tctttggagc ctttgcactg tagattctaa 2640 agaaacccaa gaattggatt ccaggcctcc atcctgaccg ttgtttcaag ggtcacatcc 2700 ccacagtctc cagccttccc cactaaaata acctggagct ctcacgggag tctgagacac 2760 ttcaggggac tacatcttcc ccagggccac tccagatgct caggggacga ctcaagccta 2820 cctagaagtt cctgcacaga gcagggtttt tgtgggtcta ggtcggacag agacctggac 2880 atgaaggagg gacagacatg ggagaggtgg ctgggaacag gggaaggttg actatttatg 2940 gagagaaaag ttaagttatt tatttataga gaatagaaag aggggaaaaa tagaaagccg 3000 tcagatgaca actaggtccc agacacaaag gtgtctcacc tcagacagga cccatctaag 3060 agagagatgg cgagagaatt agatgtgggt gaccaagggg ttctagaaga aagcacgaag 3120 ctctaaaagc cagccactgc ttggctagac atccacaggg accccctgca ccatctgtga 3180 aacccaataa acctcttttc tctgagattc tgtctgcttg tgtctgtctt gcgttggggg 3240 agaaacttcc tggtctcttt aaggagtgga gcaggggaca gaggcctcag ttggtccatg 3300 ggatccgggc agagcaaaga gacatgagga gcaggcagct cccagagaca tggtggattc 3360 acgggagtga ggcagcttaa ctgccgagag acccaaagga tgagctaggg agatccatcc 3420 aagggtggag agagatgagg gttctgggga gaagtgactc cactggaggg tgggagagtg 3480 tttaggagtg ggagggtggg ggaggggaat ccttggaaga ccggggagtc atacggattg 3540 ggagaaatcc tggaagcagg gctgtgggac ctaaatgtct gagttgatgt accgcagtca 3600 agatatggca gaggctccgt ggaaaactca cttgggagca gggacccaaa gcagcagcct 3660 gagctcatga tcagagtgaa aggagaaggc ttgtgaggtc cgtgaattcc cagggctgag 3720 ttcattccct ctgggctgcc ccatactcat cccattaccc cccccaccag ccctcccaaa 3780 gcccatgcac acttcccaac tctcaagctg ctctgccttc agccacttcc tccaagaact 3840 caaacagggg gctttccctc ctcaatatca tgtctccccc cttatgcacc cagctttcag 3900 aagcaccccc ccatgctaag ttctccccca tggatgtccc atttagaaat caaaaggaaa 3960 tagacacagg catggtcttt ctacaaagaa acagacaatg attagctctg gaggacagag 4020 aagaaatggg tttcagttct cagggtccta tacaacacac acacacacac acacacacac 4080 acacacacac acacaccctc ctgattggcc ccagattgcc acagaatcct ggtggggacg 4140 acgggggaga gattccttga tgcctgggtg tccccaactt tccaaaccct ctgcccccgc 4200 gatggagaag aaaccgagac agaggtgtag ggccactacc gcttcctcca catgagatca 4260 tggttttctc caccaaggaa gttttccgag ggttgaatga gagcttttcc ccgccctctt 4320 ccccaagggc tataaaggcg gccgtctgca cagccagcca gcagaagctc cctcagcgag 4380 gacagcaagg gactagccag gagggagaac agaaactcca gaacatcttg gaaatagctc 4440 ccagaaaagc aagcagccaa ccaggcaggt tctgtccctt tcactcactg gcccaaggcg 4500 ccacatctcc ctccagaaaa gacacc atg agc aca gaa agc atg atc cgc gac 4553 Met Ser Thr Glu Ser Met Ile Arg Asp 1 5 gtg gaa ctg gca gaa gag gca ctc ccc caa aag atg ggg ggc ttc cag 4601 Val Glu Leu Ala Glu Glu Ala Leu Pro Gln Lys Met Gly Gly Phe Gln 10 15 20 25 aac tcc agg cgg tgc cta tgt ctc agc ctc ttc tca ttc ctg ctt gtg 4649 Asn Ser Arg Arg Cys Leu Cys Leu Ser Leu Phe Ser Phe Leu Leu Val 30 35 40 gca ggg gcc acc acg ctc ttc tgt cta ctg aac ttc ggg gtg atc ggt 4697 Ala Gly Ala Thr Thr Leu Phe Cys Leu Leu Asn Phe Gly Val Ile Gly 45 50 55 ccc caa agg gat gag gtgagtgtct gggcaaccct tattctcgct cacaagcaaa 4752 Pro Gln Arg Asp Glu 60 acgggttagg agggcaagaa ggacagtgtg agggaaagaa gtgggctaat gggcagggca 4812 aggtggagga gagtgtggag gggacagagt caggacctcg gacccatgcg tccagctgac 4872 taaacatcct tcgtcggatg cacagagaga tgaatgaacg aacaagtgtg ttcacacgtg 4932 gagagatctg gaaagatgtg gccaggggaa gaggggataa gcaagagata aaactcagag 4992 acagaaatga gagaggcatg agagataagg aggaagatga aggggagata acgggagatc 5052 aagcacagag ggcaccgcag aaagaagccg tgggttggac agatgaatga atgaagaaga 5112 aaacacaaag tggggggtgg gtggggcaaa gaggaactgt aagcggggca atcagccggg 5172 agcttctcct ttggggtgag tctgtcttaa ctaacctcct tttcctacac ag aag ttc 5230 Lys Phe cca aat ggc ctc cct ctc atc agt tct atg gcc cag acc ctc aca ctc 5278 Pro Asn Gly Leu Pro Leu Ile Ser Ser Met Ala Gln Thr Leu Thr Leu 65 70 75 80 agtaagtgtt cccacacctc tctcttaatt taagatggag aagggcagtt aggcatggga 5338 Arg tgagatgggg tggggggaaa acttaaagct ttggtttggg aggaaagggg tctaagtgca 5398 tagatgcttg ctgggaagcc taaaaggctc atccttgcct ttgtctcttc ccctcca 5455 gga tca tct tct caa aat tcg agt gac aag cct gta gcc cac gtc gta 5503 Ser Ser Ser Gln Asn Ser Ser Asp Lys Pro Val Ala His Val Val 85 90 95 ggtaagattt ctttacatgt gccttgagaa tgaaggggca tgattttggg gggcgggttg 5563 aggggtgtcg agccaggctg agaaaagaca gagctcttag agacagcacg tgagagtcag 5623 agcagtgact caaaagcaag gcatcagggg gccacccggg acctcatagc caatgggatg 5683 tggaaagaca gagggtgcag gaaccggaag tgaagtgtgg gtagctgctg aggctcagga 5743 tgtggagtgt gaactaagag ggtgacactg actcaatcct cccccccccc ctca gca 5800 Ala aac cac caa gtg gag gag cag ctg gag tgg ctg agc cag cgc gcc aac 5848 Asn His Gln Val Glu Glu Gln Leu Glu Trp Leu Ser Gln Arg Ala Asn 100 105 110 gcc ctc ctg gcc aac ggc atg gat ctc aaa gac aac caa cta gtg gtg 5896 Ala Leu Leu Ala Asn Gly Met Asp Leu Lys Asp Asn Gln Leu Val Val 115 120 125 cca gcc gat ggg ttg tac ctt gtc tac tcc cag gtt ctc ttc aag gga 5944 Pro Ala Asp Gly Leu Tyr Leu Val Tyr Ser Gln Val Leu Phe Lys Gly 130 135 140 145 caa ggc tgc ccc gac tac gtg ctc ctc acc cac acc gtc agc cga ttt 5992 Gln Gly Cys Pro Asp Tyr Val Leu Leu Thr His Thr Val Ser Arg Phe 150 155 160 gct atc tca tac cag gag aaa gtc aac ctc ctc tct gcc gtc aag agc 6040 Ala Ile Ser Tyr Gln Glu Lys Val Asn Leu Leu Ser Ala Val Lys Ser 165 170 175 ccc tgc ccc aag gac acc cct gag ggg gct gag ctc aaa ccc tgg tat 6088 Pro Cys Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu Lys Pro Trp Tyr 180 185 190 gag ccc ata tac ctg gga gga gtc ttc cag ctg gag aag ggg gac caa 6136 Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Gln 195 200 205 ctc agc gct gag gtc aat ctg ccc aag tac tta gac ttt gcg gag tcc 6184 Leu Ser Ala Glu Val Asn Leu Pro Lys Tyr Leu Asp Phe Ala Glu Ser 210 215 220 225 ggg cag gtc tac ttt gga gtc att gct ctg tga agggaatggg tgttcatcca 6237 Gly Gln Val Tyr Phe Gly Val Ile Ala Leu 230 235 ttctctaccc agcccccact ctgacccctt tactctgacc cctttattgt ctactcctca 6297 gagcccccag tctgtgtcct tctaacttag aaaggggatt atggctcaga gtccaactct 6357 gtgctcagag ctttcaacaa ctactcagaa acacaagatg ctgggacagt gacctggact 6417 gtgggcctct catgcaccac catcaaggac tcaaatgggc tttccgaatt cactggagcc 6477 tcgaatgtcc attcctgagt tctgcaaagg gagagtggtc aggttgcctc tgtctcagaa 6537 tgaggctgga taagatctca ggccttccta ccttcagacc tttccagact cttccctgag 6597 gtgcaatgca cagccttcct cacagagcca gcccccctct atttatattt gcacttatta 6657 tttattattt atttattatt tatttatttg cttatgaatg tatttatttg gaaggccggg 6717 gtgtcctgga ggacccagtg tgggaagctg tcttcagaca gacatgtttt ctgtgaaaac 6777 ggagctgagc tgtccccacc tggcctctct accttgttgc ctcctctttt gcttatgttt 6837 aaaacaaaat atttatctaa cccaattgtc ttaataacgc tgatttggtg accaggctgt 6897 cgctacatca ctgaacctct gctccccacg ggagccgtga ctgtaattgc cctacagtca 6957 attgagagaa ataaagatcg cttggaaaag aaatgtgatt tctgtcttgg gatgaagtct 7017 gcatccatct ctttgcggag gcctaaagtc tctgggtcca gatctcagtc tttatacccc 7077 tgggccatta agacccccaa gacccccgtg gaacaaaagg cagccaacat ccctacctct 7137 cccccggaaa caggagccta accctaatta cctttgccct ggggcatggg aatttcccac 7197 tctgggaatt c 7208 <210> SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 108 gagcttctgc tggctggctg 20 <210> SEQ ID NO 109 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 109 ccttgctgtc ctcgctgagg 20 <210> SEQ ID NO 110 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 110 tcatggtgtc ttttctggag 20 <210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 111 ctttctgtgc tcatggtgtc 20 <210> SEQ ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 112 gcggatcatg ctttctgtgc 20 <210> SEQ ID NO 113 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 113 gggaggccat ttgggaactt 20 <210> SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 114 cgaattttga gaagatgatc 20 <210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 115 ctcctccact tggtggtttg 20 <210> SEQ ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 116 cctgagatct tatccagcct 20 <210> SEQ ID NO 117 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 117 caattacagt cacggctccc 20 <210> SEQ ID NO 118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 118 cccttcattc tcaaggcaca 20 <210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 119 cacccctcaa cccgcccccc 20 <210> SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 120 agagctctgt cttttctcag 20 <210> SEQ ID NO 121 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 121 cactgctctg actctcacgt 20 <210> SEQ ID NO 122 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 122 atgaggtccc gggtggcccc 20 <210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 123 caccctctgt ctttccacat 20 <210> SEQ ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 124 ctccacatcc tgagcctcag 20 <210> SEQ ID NO 125 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 125 attgagtcag tgtcaccctc 20 <210> SEQ ID NO 126 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 126 gctggctcag ccactccagc 20 <210> SEQ ID NO 127 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 127 tctttgagat ccatgccgtt 20 <210> SEQ ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 128 aacccatcgg ctggcaccac 20 <210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 129 gtttgagctc agccccctca 20 <210> SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 130 ctcctcccag gtatatgggc 20 <210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 131 tgagttggtc ccccttctcc 20 <210> SEQ ID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 132 caaagtagac ctgcccggac 20 <210> SEQ ID NO 133 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 133 acacccattc ccttcacaga 20 <210> SEQ ID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 134 cataatcccc tttctaagtt 20 <210> SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 135 cacagagttg gactctgagc 20 <210> SEQ ID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 136 cagcatcttg tgtttctgag 20 <210> SEQ ID NO 137 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 137 cacagtccag gtcactgtcc 20 <210> SEQ ID NO 138 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 138 tgatggtggt gcatgagagg 20 <210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 139 gtgaattcgg aaagcccatt 20 <210> SEQ ID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 140 cctgaccact ctccctttgc 20 <210> SEQ ID NO 141 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 141 tgcatccccc aggccaccat 20 <210> SEQ ID NO 142 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 142 gccgaggtcc atgtcgtacg c 21 <210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 143 tcaagcagtg ccaccgatcc 20 <210> SEQ ID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 144 agtgtcttct gtgtgccaga 20 <210> SEQ ID NO 145 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 145 gtgtcttctg tgtgccagac 20 <210> SEQ ID NO 146 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 146 tgtcttctgt gtgccagaca 20 <210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 147 gtcttctgtg tgccagacac 20 <210> SEQ ID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 148 tcttctgtgt gccagacacc 20 <210> SEQ ID NO 149 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 149 cttctgtgtg ccagacaccc 20 <210> SEQ ID NO 150 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 150 ttctgtgtgc cagacaccct 20 <210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 151 tctgtgtgcc agacacccta 20 <210> SEQ ID NO 152 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 152 ctgtgtgcca gacaccctat 20 <210> SEQ ID NO 153 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 153 tgtgtgccag acaccctatc 20 <210> SEQ ID NO 154 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 154 tgtgccagac accctatctt 20 <210> SEQ ID NO 155 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 155 gtgccagaca ccctatcttc 20 <210> SEQ ID NO 156 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 156 tgccagacac cctatcttct 20 <210> SEQ ID NO 157 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 157 gccagacacc ctatcttctt 20 <210> SEQ ID NO 158 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 158 ccagacaccc tatcttcttc 20 <210> SEQ ID NO 159 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 159 cagacaccct atcttcttct 20 <210> SEQ ID NO 160 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 160 agacacccta tcttcttctc 20 <210> SEQ ID NO 161 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 161 gacaccctat cttcttctct 20 <210> SEQ ID NO 162 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 162 acaccctatc ttcttctctc 0 <210> SEQ ID NO 163 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 163 caccctatct tcttctctcc 20 <210> SEQ ID NO 164 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 164 gtcttctgtg tgccagac 18 <210> SEQ ID NO 165 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 165 tcttctgtgt gccagaca 18 <210> SEQ ID NO 166 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 166 cttctgtgtg ccagacac 18 <210> SEQ ID NO 167 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 167 ttctgtgtgc cagacacc 18 <210> SEQ ID NO 168 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 168 tctgtgtgcc agacaccc 18 <210> SEQ ID NO 169 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 169 ctgtgtgcca gacaccct 18 <210> SEQ ID NO 170 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 170 tgtgtgccag acacccta 18 <210> SEQ ID NO 171 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 171 gtgtgccaga caccctat 18 <210> SEQ ID NO 172 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 172 tgtgccagac accctatc 18 <210> SEQ ID NO 173 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 173 tgccagacac cctatctt 18 <210> SEQ ID NO 174 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 174 gccagacacc ctatcttc 18 <210> SEQ ID NO 175 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 175 ccagacaccc tatcttct 18 <210> SEQ ID NO 176 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 176 cagacaccct atcttctt 18 <210> SEQ ID NO 177 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 177 agacacccta tcttcttc 18 <210> SEQ ID NO 178 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 178 gacaccctat cttcttct 18 <210> SEQ ID NO 179 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 179 acaccctatc ttcttctc 18 <210> SEQ ID NO 180 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 180 agaggtttgg agacacttac 20 <210> SEQ ID NO 181 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 181 gaattaggaa agaggtttgg 20 <210> SEQ ID NO 182 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 182 cccaaaccca gaattaggaa 20 <210> SEQ ID NO 183 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 183 tacccccaaa cccaaaccca 20 <210> SEQ ID NO 184 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 184 gtactaaccc tacccccaaa 20 <210> SEQ ID NO 185 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 185 ttccataccg gtactaaccc 20 <210> SEQ ID NO 186 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 186 cccccactgc ttccataccg 20 <210> SEQ ID NO 187 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 187 ctttaaattt cccccactgc 20 <210> SEQ ID NO 188 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 188 aagaccaaaa ctttaaattt 20 <210> SEQ ID NO 189 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 189 atcctccccc aagaccaaaa 20 <210> SEQ ID NO 190 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 190 acctccatcc atcctccccc 20 <210> SEQ ID NO 191 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 191 ccctactttc acctccatcc 20 <210> SEQ ID NO 192 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 192 gaaaataccc ccctactttc 20 <210> SEQ ID NO 193 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 193 aaacttccta gaaaataccc 20 <210> SEQ ID NO 194 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 194 tgagaccctt aaacttccta 20 <210> SEQ ID NO 195 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 195 aagaaaaagc tgagaccctt 20 <210> SEQ ID NO 196 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 196 ggagagagaa aagaaaaagc 20 <210> SEQ ID NO 197 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 197 tgagccagaa gaggttgagg 20 <210> SEQ ID NO 198 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 198 attctctttt tgagccagaa 20 <210> SEQ ID NO 199 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 199 taagccccca attctctttt 20 <210> SEQ ID NO 200 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 200 gttccgaccc taagccccca 20 <210> SEQ ID NO 201 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 201 ctaagcttgg gttccgaccc 20 <210> SEQ ID NO 202 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 202 gcttaaagtt ctaagcttgg 20 <210> SEQ ID NO 203 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 203 tggtcttgtt gcttaaagtt 20 <210> SEQ ID NO 204 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 204 ttcgaagtgg tggtcttgtt 20 <210> SEQ ID NO 205 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 205 aatcccaggt ttcgaagtgg 20 <210> SEQ ID NO 206 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 206 cacattcctg aatcccaggt 20 <210> SEQ ID NO 207 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 207 gtgcaggcca cacattcctg 20 <210> SEQ ID NO 208 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 208 gcacttcact gtgcaggcca 20 <210> SEQ ID NO 209 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 209 gtggttgcca gcacttcact 20 <210> SEQ ID NO 210 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 210 tgaattctta gtggttgcca 20 <210> SEQ ID NO 211 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 211 ggccccagtt tgaattctta 20 <210> SEQ ID NO 212 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 212 gagttctgga ggccccagtt 20 <210> SEQ ID NO 213 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 213 aggccccagt gagttctgga 20 <210> SEQ ID NO 214 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 214 tcaaagctgt aggccccagt 20 <210> SEQ ID NO 215 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 215 atgtcaggga tcaaagctgt 20 <210> SEQ ID NO 216 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 216 cagattccag atgtcaggga 20 <210> SEQ ID NO 217 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 217 ccctggtctc cagattccag 20 <210> SEQ ID NO 218 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 218 accaaaggct ccctggtctc 20 <210> SEQ ID NO 219 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 219 tctggccaga accaaaggct 20 <210> SEQ ID NO 220 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 220 cctgcagcat tctggccaga 20 <210> SEQ ID NO 221 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 221 cttctcaagt cctgcagcat 20 <210> SEQ ID NO 222 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 222 taggtgaggt cttctcaagt 20 <210> SEQ ID NO 223 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 223 tgtcaatttc taggtgaggt 20 <210> SEQ ID NO 224 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 224 ggtccacttg tgtcaatttc 20 <210> SEQ ID NO 225 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 225 gaaggcctaa ggtccacttg 20 <210> SEQ ID NO 226 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 226 ctggagagag gaaggcctaa 20 <210> SEQ ID NO 227 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 227 ctggaaacat ctggagagag 20 <210> SEQ ID NO 228 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 228 tcaaggaagt ctggaaacat 20 <210> SEQ ID NO 229 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 229 gctccgtgtc tcaaggaagt 20 <210> SEQ ID NO 230 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 230 ataaatacat tcatctgtaa 20 <210> SEQ ID NO 231 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 231 ggtctcccaa ataaatacat 20 <210> SEQ ID NO 232 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 232 aggatacccc ggtctcccaa 20 <210> SEQ ID NO 233 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 233 tgggtccccc aggatacccc 20 <210> SEQ ID NO 234 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 234 gctcctacat tgggtccccc 20 <210> SEQ ID NO 235 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 235 agccaaggca gctcctacat 20 <210> SEQ ID NO 236 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 236 aacatgtctg agccaaggca 20 <210> SEQ ID NO 237 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 237 tttcacggaa aacatgtctg 20 <210> SEQ ID NO 238 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 238 tcagctccgt tttcacggaa 20 <210> SEQ ID NO 239 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 239 agcctattgt tcagctccgt 20 <210> SEQ ID NO 240 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 240 acatgggaac agcctattgt 20 <210> SEQ ID NO 241 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 241 atcaaaagaa ggcacagagg 20 <210> SEQ ID NO 242 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 242 gtttagacaa cttaatcaga 20 <210> SEQ ID NO 243 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 243 aatcagcatt gtttagacaa 20 <210> SEQ ID NO 244 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 244 ttggtcacca aatcagcatt 20 <210> SEQ ID NO 245 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 245 tgagtgacag ttggtcacca 20 <210> SEQ ID NO 246 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 246 ggctcagcaa tgagtgacag 20 <210> SEQ ID NO 247 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 247 attacagaca caactcccct 20 <210> SEQ ID NO 248 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 248 tagtagggcg attacagaca 20 <210> SEQ ID NO 249 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 249 cgccactgaa tagtagggcg 20 <210> SEQ ID NO 250 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 250 ctttatttct cgccactgaa 20 <210> SEQ ID NO 251 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 251 ctgagggagc gtctgctggc 20 <210> SEQ ID NO 252 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 252 ccttgctgag ggagcgtctg 20 <210> SEQ ID NO 253 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 253 ctggtcctct gctgtccttg 20 <210> SEQ ID NO 254 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 254 cctctgctgt ccttgctgag 20 <210> SEQ ID NO 255 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 255 ttctctccct cttagctggt 20 <210> SEQ ID NO 256 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 256 tccctcttag ctggtcctct 20 <210> SEQ ID NO 257 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 257 tctgagggtt gttttcaggg 20 <210> SEQ ID NO 258 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 258 ctgtagttgc ttctctccct 20 <210> SEQ ID NO 259 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 259 acctgcctgg cagcttgtca 20 <210> SEQ ID NO 260 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 260 ggatgtggcg tctgagggtt 20 <210> SEQ ID NO 261 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 261 tgtgagagga agagaacctg 20 <210> SEQ ID NO 262 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 262 gaggaagaga acctgcctgg 20 <210> SEQ ID NO 263 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 263 agccgtgggt cagtatgtga 20 <210> SEQ ID NO 264 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 264 tgggtcagta tgtgagagga 20 <210> SEQ ID NO 265 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 265 gagagggtga agccgtgggt 20 <210> SEQ ID NO 266 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 266 tcatggtgtc ctttccaggg 20 <210> SEQ ID NO 267 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 267 ctttcagtgc tcatggtgtc 20 <210> SEQ ID NO 268 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 268 tcatgctttc agtgctcatg 20 <210> SEQ ID NO 269 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 269 acgtcccgga tcatgctttc 20 <210> SEQ ID NO 270 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 270 gctccacgtc ccggatcatg 20 <210> SEQ ID NO 271 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 271 tcctcggcca gctccacgtc 20 <210> SEQ ID NO 272 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 272 gcgcctcctc ggccagctcc 20 <210> SEQ ID NO 273 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 273 aggaacaagc accgcctgga 20 <210> SEQ ID NO 274 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 274 caagcaccgc ctggagccct 20 <210> SEQ ID NO 275 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 275 aaggagaaga ggctgaggaa 20 <210> SEQ ID NO 276 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 276 gaagaggctg aggaacaagc 20 <210> SEQ ID NO 277 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 277 cctgccacga tcaggaagga 20 <210> SEQ ID NO 278 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 278 cacgatcagg aaggagaaga 20 <210> SEQ ID NO 279 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 279 aagagcgtgg tggcgcctgc 20 <210> SEQ ID NO 280 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 280 cgtggtggcg cctgccacga 20 <210> SEQ ID NO 281 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 281 aagtgcagca ggcagaagag 20 <210> SEQ ID NO 282 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 282 cagcaggcag aagagcgtgg 20 <210> SEQ ID NO 283 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 283 gatcactcca aagtgcagca 20 <210> SEQ ID NO 284 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 284 gggccgatca ctccaaagtg 20 <210> SEQ ID NO 285 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 285 gggccagagg gctgattaga 20 <210> SEQ ID NO 286 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 286 agagggctga ttagagagag 20 <210> SEQ ID NO 287 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 287 gctacaggct tgtcactcgg 20 <210> SEQ ID NO 288 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 288 ctgactgcct gggccagagg 20 <210> SEQ ID NO 289 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 289 tacaacatgg gctacaggct 20 <210> SEQ ID NO 290 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 290 agccactgga gctgcccctc 20 <210> SEQ ID NO 291 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 291 ctggagctgc ccctcagctt 20 <210> SEQ ID NO 292 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 292 ttggcccggc ggttcagcca 20 <210> SEQ ID NO 293 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 293 ttggccagga gggcattggc 20 <210> SEQ ID NO 294 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 294 ccggcggttc agccactgga 20 <210> SEQ ID NO 295 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 295 ctcagctcca cgccattggc 20 <210> SEQ ID NO 296 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 296 caggagggca ttggcccggc 20 <210> SEQ ID NO 297 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 297 ctccacgcca ttggccagga 20 <210> SEQ ID NO 298 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 298 accagctggt tatctctcag 20 <210> SEQ ID NO 299 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 299 ctggttatct ctcagctcca 20 <210> SEQ ID NO 300 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 300 ccctctgatg gcaccaccag 20 <210> SEQ ID NO 301 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 301 tgatggcacc accagctggt 20 <210> SEQ ID NO 302 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 302 tagatgaggt acaggccctc 20 <210> SEQ ID NO 303 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 303 aagaggacct gggagtagat 20 <210> SEQ ID NO 304 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 304 gaggtacagg ccctctgatg 20 <210> SEQ ID NO 305 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 305 cagccttggc ccttgaagag 20 <210> SEQ ID NO 306 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 306 gacctgggag tagatgaggt 20 <210> SEQ ID NO 307 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 307 ttggcccttg aagaggacct 20 <210> SEQ ID NO 308 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 308 tggtgtgggt gaggagcaca 20 <210> SEQ ID NO 309 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 309 cggcgatgcg gctgatggtg 20 <210> SEQ ID NO 310 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 310 tgggtgagga gcacatgggt 20 <210> SEQ ID NO 311 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 311 tggtctggta ggagacggcg 20 <210> SEQ ID NO 312 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 312 atgcggctga tggtgtgggt 20 <210> SEQ ID NO 313 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 313 agaggaggtt gaccttggtc 20 <210> SEQ ID NO 314 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 314 tggtaggagac ggcgatgcg 20 <210> SEQ ID NO 315 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 315 aggttgacct tggtctggta 20 <210> SEQ ID NO 316 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 316 ggctcttgat ggcagagagg 20 <210> SEQ ID NO 317 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 317 tcataccaggg cttggcctc 20 <210> SEQ ID NO 318 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 318 ttgatggcag agaggaggtt 20 <210> SEQ ID NO 319 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 319 agctggaaga cccctcccag 20 <210> SEQ ID NO 320 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 320 atagatgggc tcataccagg 20 <210> SEQ ID NO 321 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 321 cggtcaccct tctccagctg 20 <210> SEQ ID NO 322 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 322 gaagacccct cccagataga 20 <210> SEQ ID NO 323 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 323 acccttctcc agctggaaga 20 <210> SEQ ID NO 324 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 324 tcggcaaagt cgagatagtc 20 <210> SEQ ID NO 325 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 325 gggccgattg atctcagcgc 20 <210> SEQ ID NO 326 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 326 tagacctgcc cagactcggc 20 <210> SEQ ID NO 327 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 327 aaagtcgaga tagtcgggcc 20 <210> SEQ ID NO 328 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 328 gcaatgatcc caaagtagac 20 <210> SEQ ID NO 329 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 329 ctgcccagac tcggcaaagt 20 <210> SEQ ID NO 330 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 330 cgtcctcctc acagggcaat 20 <210> SEQ ID NO 331 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 331 ggaaggttgg atgttcgtcc 20 <210> SEQ ID NO 332 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 332 tcctcacagg gcaatgatcc 20 <210> SEQ ID NO 333 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 333 gttgagggtg tctgaaggag 20 <210> SEQ ID NO 334 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 334 gttggatgtt cgtcctcctc 20 <210> SEQ ID NO 335 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 335 tttgagccag aagaggttga 20 <210> SEQ ID NO 336 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 336 gaggcgtttg ggaaggttgg 20 <210> SEQ ID NO 337 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 337 gcccccaatt ctctttttga 20 <210> SEQ ID NO 338 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 338 gccagaagag gttgagggtg 20 <210> SEQ ID NO 339 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 339 gggttccgac cctaagcccc 20 <210> SEQ ID NO 340 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 340 caattctctt tttgagccag 20 <210> SEQ ID NO 341 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 341 taaagttcta agcttgggtt 20 <210> SEQ ID NO 342 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 342 ccgaccctaa gcccccaatt 20 <210> SEQ ID NO 343 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 343 ggtggtcttg ttgcttaaag 20 <210> SEQ ID NO 344 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 344 ttctaagctt gggttccgac 20 <210> SEQ ID NO 345 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 345 cccaggtttc gaagtggtgg 20 <210> SEQ ID NO 346 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 346 tcttgttgct taaagttcta 20 <210> SEQ ID NO 347 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 347 cacacattcc tgaatcccag 20 <210> SEQ ID NO 348 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 348 gtttcgaagt ggtggtcttg 20 <210> SEQ ID NO 349 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 349 cttcactgtg caggccacac 20 <210> SEQ ID NO 350 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 350 attcctgaat cccaggtttc 20 <210> SEQ ID NO 351 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 351 tagtggttgc cagcacttca 20 <210> SEQ ID NO 352 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 352 cccagtttga attcttagtg 20 <210> SEQ ID NO 353 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 353 ctgtgcaggc cacacattcc 20 <210> SEQ ID NO 354 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 354 gtgagttctg gaggccccag 20 <210> SEQ ID NO 355 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 355 gttgccagca cttcactgtg 20 <210> SEQ ID NO 356 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 356 tttgaattct tagtggttgc 20 <210> SEQ ID NO 357 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 357 aagctgtagg ccccagtgag 20 <210> SEQ ID NO 358 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 358 ttctggaggc cccagtttga 20 <210> SEQ ID NO 359 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 359 agatgtcagg gatcaaagct 20 <210> SEQ ID NO 360 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 360 tggtctccag attccagatg 20 <210> SEQ ID NO 361 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 361 gtaggcccca gtgagttctg 20 <210> SEQ ID NO 362 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 362 gaaccaaagg ctccctggtc 20 <210> SEQ ID NO 363 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 363 tcagggatca aagctgtagg 20 <210> SEQ ID NO 364 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 364 tccagattcc agatgtcagg 20 <210> SEQ ID NO 365 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 365 gcagcattct ggccagaacc 20 <210> SEQ ID NO 366 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 366 gtcttctcaa gtcctgcagc 20 <210> SEQ ID NO 367 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 367 aaaggctccc tggtctccag 20 <210> SEQ ID NO 368 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 368 caatttctag gtgaggtctt 20 <210> SEQ ID NO 369 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 369 attctggcca gaaccaaagg 20 <210> SEQ ID NO 370 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 370 aaggtccact tgtgtcaatt 20 <210> SEQ ID NO 371 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 371 gagagaggaa ggcctaaggt 20 <210> SEQ ID NO 372 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 372 tctaggtgag gtcttctcaa 20 <210> SEQ ID NO 373 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 373 ccacttgtgt caatttctag 20 <210> SEQ ID NO 374 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 374 gtctggaaac atctggagag 20 <210> SEQ ID NO 375 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 375 ccgtgtctca aggaagtctg 20 <210> SEQ ID NO 376 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 376 aggaaggcct aaggtccact 20 <210> SEQ ID NO 377 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 377 gagggagctg gctccatggg 20 <210> SEQ ID NO 378 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 378 gaaacatctg gagagaggaa 20 <210> SEQ ID NO 379 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 379 gtgcaaacat aaatagaggg 20 <210> SEQ ID NO 380 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 380 tctcaaggaa gtctggaaac 20 <210> SEQ ID NO 381 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 381 aataaataat cacaagtgca 20 <210> SEQ ID NO 382 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 382 gggctgggct ccgtgtctca 20 <210> SEQ ID NO 383 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 383 taccccggtc tcccaaataa 20 <210> SEQ ID NO 384 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 384 aacataaata gagggagctg 20 <210> SEQ ID NO 385 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 385 ttgggtcccc caggataccc 20 <210> SEQ ID NO 386 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 386 ataatcacaa gtgcaaacat 20 <210> SEQ ID NO 387 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 387 aaggcagctc ctacattggg 20 <210> SEQ ID NO 388 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 388 cggtctccca aataaataca 20 <210> SEQ ID NO 389 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 389 aaacatgtct gagccaaggc 20 <210> SEQ ID NO 390 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 390 tcccccagga taccccggtc 20 <210> SEQ ID NO 391 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 391 agctcctaca ttgggtcccc 20 <210> SEQ ID NO 392 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 392 tgtctgagcc aaggcagctc 20 <210> SEQ ID NO 393 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 393 cagcctattg ttcagctccg 20 <210> SEQ ID NO 394 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 394 agaaggcaca gaggccaggg 20 <210> SEQ ID NO 395 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 395 ttttcacgga aaacatgtct 20 <210> SEQ ID NO 396 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 396 tattgttcag ctccgttttc 20 <210> SEQ ID NO 397 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 397 aaaaacataa tcaaaagaag 20 <210> SEQ ID NO 398 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 398 cagataaata ttttaaaaaa 20 <210> SEQ ID NO 399 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 399 tacatgggaa cagcctattg 20 <210> SEQ ID NO 400 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 400 tttagacaac ttaatcagat 20 <210> SEQ ID NO 401 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 401 cataatcaaa agaaggcaca 20 <210> SEQ ID NO 402 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 402 accaaatcag cattgtttag 20 <210> SEQ ID NO 403 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 403 aaatatttta aaaaacataa 20 <210> SEQ ID NO 404 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 404 gagtgacagt tggtcaccaa 20 <210> SEQ ID NO 405 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 405 acaacttaatc agataaata 20 <210> SEQ ID NO 406 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 406 cagaggctca gcaatgagtg 20 <210> SEQ ID NO 407 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 407 atcagcattg tttagacaac 20 <210> SEQ ID NO 408 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 408 agggcgatta cagacacaac 20 <210> SEQ ID NO 409 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 409 acagttggtc accaaatcag 20 <210> SEQ ID NO 410 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 410 tcgccactga atagtagggc 20 <210> SEQ ID NO 411 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 411 gctcagcaat gagtgacagt 20 <210> SEQ ID NO 412 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 412 agcaaacttt atttctcgcc 20 <210> SEQ ID NO 413 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 413 gattacagac acaactcccc 20 <210> SEQ ID NO 414 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 414 actgaatagt agggcgatta 20 <210> SEQ ID NO 415 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 415 actttatttc tcgccactga 20 <210> SEQ ID NO 416 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 416 gctgtccttg ctgagggagc 20 <210> SEQ ID NO 417 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 417 cttagctggt cctctgctgt 20 <210> SEQ ID NO 418 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 418 gttgcttctc tccctcttag 20 <210> SEQ ID NO 419 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 419 tggcgtctga gggttgtttt 20 <210> SEQ ID NO 420 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 420 agagaacctg cctggcagct 20 <210> SEQ ID NO 421 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 421 cagtatgtga gaggaagaga 20 <210> SEQ ID NO 422 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 422 ggtgaagccg tgggtcagta 20 <210> SEQ ID NO 423 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 423 agtgctcatg gtgtcctttc 20 <210> SEQ ID NO 424 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 424 ccggatcatg ctttcagtgc 20 <210> SEQ ID NO 425 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 425 ggccagctcc acgtcccgga 20 <210> SEQ ID NO 426 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 426 ggcccccctg tcttcttggg 20 <210> SEQ ID NO 427 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 427 ggctgaggaa caagcaccgc 20 <210> SEQ ID NO 428 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 428 tcaggaagga gaagaggctg 20 <210> SEQ ID NO 429 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 429 tggcgcctgc cacgatcagg 20 <210> SEQ ID NO 430 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 430 ggcagaagag cgtggtggcg 20 <210> SEQ ID NO 431 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 431 ctccaaagtg cagcaggcag 20 <210> SEQ ID NO 432 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 432 gctgattaga gagaggtccc 20 <210> SEQ ID NO 433 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 433 tgcctgggcc agagggctga 20 <210> SEQ ID NO 434 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 434 gctgcccctc agcttgaggg 20 <210> SEQ ID NO 435 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 435 ggttcagcca ctggagctgc 20 <210> SEQ ID NO 436 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 436 gggcattggc ccggcggttc 20 <210> SEQ ID NO 437 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 437 cgccattggc caggagggca 20 <210> SEQ ID NO 438 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 438 tatctctcag ctccacgcca 20 <210> SEQ ID NO 439 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 439 gcaccaccag ctggttatct 20 <210> SEQ ID NO 440 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 440 acaggccctc tgatggcacc 20 <210> SEQ ID NO 441 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 441 gggagtagat gaggtacagg 20 <210> SEQ ID NO 442 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 442 ccttgaagag gacctgggag 20 <210> SEQ ID NO 443 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 443 gaggagcaca tgggtggagg 20 <210> SEQ ID NO 444 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 444 gctgatggtg tgggtgagga 20 <210> SEQ ID NO 445 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 445 ggagacggcg atgcggctga 20 <210> SEQ ID NO 446 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 446 gaccttggtc tggtaggaga 20 <210> SEQ ID NO 447 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 447 ggcagagagg aggttgacct 20 <210> SEQ ID NO 448 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 448 tgggctcata ccagggcttg 20 <210> SEQ ID NO 449 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 449 cccctcccag atagatgggc 20 <210> SEQ ID NO 450 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 450 tgagtcggtc acccttctcc 20 <210> SEQ ID NO 451 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 451 gattgatctc agcgctgagt 20 <210> SEQ ID NO 452 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 452 cgagatagtc gggccgattg 20 <210> SEQ ID NO 453 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 453 caaagtagac ctgcccagac 20 <210> SEQ ID NO 454 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 454 acagggcaat gatcccaaag 20 <210> SEQ ID NO 455 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 455 atgttcgtcc tcctcacagg 20 <210> SEQ ID NO 456 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 456 gtttgggaag gttggatgtt 20 <210> SEQ ID NO 457 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 457 aagaggttga gggtgtctga 20 <210> SEQ ID NO 458 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 458 ctctttttga gccagaagag 20 <210> SEQ ID NO 459 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 459 cctaagcccc caattctctt 20 <210> SEQ ID NO 460 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 460 agcttgggtt ccgaccctaa 20 <210> SEQ ID NO 461 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 461 ttgcttaaag ttctaagctt 20 <210> SEQ ID NO 462 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 462 gaagtggtgg tcttgttgct 20 <210> SEQ ID NO 463 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 463 tgaatcccag gtttcgaagt 20 <210> SEQ ID NO 464 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 464 caggccacac attcctgaat 20 <210> SEQ ID NO 465 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 465 cagcacttca ctgtgcaggc 20 <210> SEQ ID NO 466 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 466 attcttagtg gttgccagca 20 <210> SEQ ID NO 467 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 467 gaggccccag tttgaattct 20 <210> SEQ ID NO 468 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 468 ccccagtgag ttctggaggc 20 <210> SEQ ID NO 469 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 469 gatcaaagct gtaggcccca 0 <210> SEQ ID NO 470 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 470 attccagatg tcagggatca 20 <210> SEQ ID NO 471 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 471 ctccctggtc tccagattcc 20 <210> SEQ ID NO 472 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 472 ggccagaacc aaaggctccc 20 <210> SEQ ID NO 473 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 473 gtcctgcagc attctggcca 20 <210> SEQ ID NO 474 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 474 gtgaggtctt ctcaagtcct 20 <210> SEQ ID NO 475 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 475 tgtgtcaatt tctaggtgag 20 <210> SEQ ID NO 476 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 476 ggcctaaggt ccacttgtgt 20 <210> SEQ ID NO 477 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 477 atctggagag aggaaggcct 20 <210> SEQ ID NO 478 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 478 aggaagtctg gaaacatctg 20 <210> SEQ ID NO 479 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 479 gggctccgtg tctcaaggaa 20 <210> SEQ ID NO 480 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 480 aaatagaggg agctggctcc 20 <210> SEQ ID NO 481 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 481 cacaagtgca aacataaata 20 <210> SEQ ID NO 482 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 482 tcccaaataa atacattcat 20 <210> SEQ ID NO 483 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 483 caggataccc cggtctccca 20 <210> SEQ ID NO 484 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 484 ctacattggg tcccccagga 20 <210> SEQ ID NO 485 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 485 gagccaaggc agctcctaca 20 <210> SEQ ID NO 486 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 486 acggaaaaca tgtctgagcc 20 <210> SEQ ID NO 487 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 487 ttcagctccg ttttcacgga 20 <210> SEQ ID NO 488 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 488 gggaacagcc tattgttcag 20 <210> SEQ ID NO 489 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 489 tcaaaagaag gcacagaggc 20 <210> SEQ ID NO 490 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 490 ttttaaaaaa cataatcaaa 20 <210> SEQ ID NO 491 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 491 ttaatcagat aaatatttta 20 <210> SEQ ID NO 492 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 492 cattgtttag acaacttaat 20 <210> SEQ ID NO 493 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 493 tggtcaccaa atcagcattg 20 <210> SEQ ID NO 494 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 494 gcaatgagtg acagttggtc 20 <210> SEQ ID NO 495 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 495 gggagcagag gctcagcaat 20 <210> SEQ ID NO 496 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 496 atagtagggc gattacagac 20 <210> SEQ ID NO 497 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 497 atttctcgcc actgaatagt 20 <210> SEQ ID NO 498 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 498 ctgattagag agaggtccc 19 <210> SEQ ID NO 499 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 499 ctgattagag agaggtcc 18 <210> SEQ ID NO 500 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 500 tgagtgtctt ctgtgtgcca 20 <210> SEQ ID NO 501 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 501 gagtgtcttc tgtgtgccag 20 <210> SEQ ID NO 502 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 502 caccaagctg cggtccccaa 20 <210> SEQ ID NO 503 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 503 tccgtcatcg ctcctcaggg 20 ?? 1 Error Main Document Only. 

What is claimed is:
 1. An double stranded RNA compound between about 8 and 80 nucleobases in length targeted to a nucleic acid molecule encoding human TNF-α, wherein said compound specifically hybridizes with said nucleic acid molecule encoding TNF-α and inhibits the expression of survivin.
 2. The compound of claim 1 comprising between about 12 and 50 nucleobases in length.
 3. The compound of claim 2 comprising between about 15 and 30 nucleobases in length.
 4. The compound of claim 1, wherein said compound comprises SEQ ID NO:432.
 5. The compound of claim 1 wherein the sugar backbone is replaced with an amide-containing backbone.
 6. The compound of claim 1, wherein said amide-containing backbone is an aminoethylglycine backbone.
 7. A double stranded RNA compound having SEQ ID NO:
 432. 